53014, a human metalloprotease family member and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated 53014 nucleic acid molecules, which encode novel metalloprotease family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 53014 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 53014 gene has been introduced or disrupted. The invention still further provides isolated 53014 proteins, fusion proteins, antigenic peptides and anti-53014 antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/258,373, filed Dec. 22, 2000, the contents of whichare incorporated herein by this reference.

BACKGROUND OF THE INVENTION

[0002] Metalloproteases (also referred to herein as metallopeptidases ormetalloproteinases) are a group of highly diverse, widely distributedproteolytic enzymes that depend on bound Ca²⁺ or Zn²⁺ for activity.Certain metalloproteases can readily utilize Mn²⁺ and Mg²⁺ as well.About 30 families of metalloproteases are recognized. The mostthoroughly characterized of the metalloproteases is thermolysin, amember of the M4 metalloprotease family. In general, the biologicalfunctions of metalloproteases include protein maturation, degradation ofproteins, such as extracellular matrix proteins, tumor growth,metastasis and angiogenesis, among others.

[0003] Another metalloprotease family, the M12 family, contains thereprolysin (M12B) subfamily, which contains the snake venommetalloproteases and adamalysins (ADAMs) family. The known members ofthe reprolysin subfamily mostly lack essential peptidase active sites,but typically contain C-terminal disintegrin-like, EGF-like, andtransmembrane domains (Rawlings et al. (1995) Meth. Enzymol.248:183-228). These include BRCA1, a human breast cancer-associatedprotein, and mammalian fertilin.

[0004] ADAMs comprise a broad family of multifunctional proteins,characterized as having a disintegrin and metalloprotease domain(Wolfsberg et al. (1995) Developmental Biol. 169:378-383; Wolfsberg etal. (1995) J. Cell Biol. 131:275-278; Hurskainen et al. (1999) J. Biol.Chem. 274:25555-25563). Most ADAMs family members are similar in domainorganization, having from amino to carboxyl termini, a signal peptide, aproregion, a zinc-metalloprotease catalytic domain of the reprolysintype, a disintegrin domain, a cysteine-rich domain, an epidermal growthfactor-like domain, and in many cases a membrane-spanning region and acytoplasmic domain with signaling potential. Members of the ADAMs familyof proteins include, but are not limited to, MDC (ADAM1), ADAMTS-1,fertilin (ADAM2), cryitestin (ADAM3), epididymal apical protein I,meltrin, MS2, TNF-a converting enzyme, Kuzbanian and metargidin.ADAMTS-1 differs from other ADAM family members due to a lack of thecysteine rich, EGF and transmembrane domains and the addition ofthrombospondin type I motifs (Kuno et al. (1997) J. Biol. Chem.272:556-562).

[0005] Collagens are extracellular matrix proteins responsible for thearchitecture and structural integrity of most tissues. They aresynthesized as procollagens and go through a series ofpost-translational modifications both inside and outside the cell beforethey are fully functional (reviewed in Duance and Bailey (1981) inHandbook of Inflammation. Vol. 3 Tissue Regeneration and Repair, ed.Glynn, LE Elsevier, Amsterdam, 51-109). Included in collagen maturationare the steps of removing the extension peptides from the N- andC-termini. The failure of this process results in many connective tissuedisorders, for example, Ehlers-Danlos syndrome type VIIC, characterizedby the retention of the N-terminal propeptide of collagen I (Lenaers etal. (1971) Eur. J. Biochem. 23:533-543) by a metalloprotease,procollagen I N-protease (Colige et al. (1999) Am. J. Hum. Genet.65:308-317). This is an M12, reprolysin subfamily member with nodisintegrin-like domain, but with four thrombospondin type I motifs.

[0006] Thus, metalloproteases are likely to play important roles in awide range of diseases including, but not limited to, cancer, arthritis,Alzheimer's disease, and a variety of inflammatory conditions.Accordingly, metalloproteases are an important target for drug actionand development. Therefore, it is valuable to the field ofpharmaceutical development to identify and characterize previouslyunknown metalloproteases.

SUMMARY OF THE INVENTION

[0007] The present invention is based, in part, on the discovery of anovel metalloprotease family, e.g. the reprolysin (M12B) family (e.g.the ADAMTS (A disintegrin and metalloproteinase with thrombospondinmotifs) family) member, referred to herein as “53014”. The nucleotidesequences of cDNAs encoding 53014 are shown in SEQ ID NOs: 1 and 4, andthe amino acid sequences of 53014 polypeptides are shown in SEQ ID NOs:2 and 5. In addition, the nucleotide sequences of the coding regions aredepicted in SEQ ID NOs: 3 and 6.

[0008] Accordingly, in one aspect, the invention features a nucleic acidmolecule which encodes a 53014 protein or polypeptide, e.g., abiologically active portion of the 53014 protein. In a preferredembodiment, the isolated nucleic acid molecule encodes a polypeptidehaving the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5. In otherembodiments, the invention provides isolated 53014 nucleic acidmolecules having the nucleotide sequence shown in SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number ______. Instill other embodiments, the invention provides nucleic acid moleculesthat are substantially identical (e.g., naturally occurring allelicvariants) to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6 or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC Accession Number ______. Inother embodiments, the invention provides a nucleic acid molecule whichhybridizes under a stringent hybridization condition as described hereinto a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6 or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC AccessionNumber ______, wherein the nucleic acid encodes a full length 53014protein or an active fragment thereof.

[0009] In a related aspect, the invention further provides nucleic acidconstructs which include a 53014 nucleic acid molecule described herein.In certain embodiments, the nucleic acid molecules of the invention areoperatively linked to native or heterologous regulatory sequences. Alsoincluded are vectors and host cells containing the 53014 nucleic acidmolecules of the invention e.g., vectors and host cells suitable forproducing polypeptides.

[0010] In another related aspect, the invention provides nucleic acidfragments suitable as primers or hybridization probes for the detectionof 53014-encoding nucleic acids.

[0011] In still another related aspect, isolated nucleic acid moleculesthat are antisense to a 53014 encoding nucleic acid molecule areprovided.

[0012] In another aspect, the invention features 53014 polypeptides, andbiologically active or antigenic fragments thereof that are useful,e.g., as reagents or targets in assays applicable to treatment anddiagnosis of metalloprotease-associated or other 53014-associateddisorders. In another embodiment, the invention provides 53014polypeptides having a 53014 activity. Preferred polypeptides are 53014proteins including at least one reprolysin domain, at least onereprolysin family propeptide domain, at least one, two, three,preferably four thrombospondin domains, and, preferably, having a 53014activity, e.g., a 53014 activity as described herein (e.g., a zincbinding activity or a metalloprotease activity).

[0013] In other embodiments, the invention provides 53014 polypeptides,e.g., a 53014 polypeptide having the amino acid sequence shown in SEQ IDNO: 2, SEQ ID NO: 5 or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with ATCC Accession Number ______; anamino acid sequence that is substantially identical to the amino acidsequence shown in SEQ ID NO: 2, SEQ ID NO: 5 or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with ATCC AccessionNumber ______; or an amino acid sequence encoded by a nucleic acidmolecule having a nucleotide sequence which hybridizes under a stringenthybridization condition as described herein to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 6 or the nucleotide sequence of the insert of theplasmid deposited with ATCC Accession Number ______, wherein the nucleicacid encodes a full length 53014 protein or an active fragment thereof.

[0014] In a related aspect, the invention further provides nucleic acidconstructs which include a 53014 nucleic acid molecule described herein.

[0015] In a related aspect, the invention provides 53014 polypeptides orfragments operatively linked to non-53014 polypeptides to form fusionproteins.

[0016] In another aspect, the invention features antibodies andantigen-binding fragments thereof, that react with, or more preferablyspecifically or selectively bind 53014 polypeptides.

[0017] In another aspect, the invention provides methods of screeningfor compounds that modulate the expression or activity of the 53014polypeptides or nucleic acids.

[0018] In still another aspect, the invention provides a process formodulating 53014 polypeptide or nucleic acid expression or activity,e.g., using the compounds identified in the screens described herein. Incertain embodiments, the methods involve treatment of conditions relatedto aberrant activity or expression of the 53014 polypeptides or nucleicacids, such as conditions or disorders involving aberrant or deficientmetalloprotease, e.g. reprolysin (M12B) (e.g. ADAMTS) function orexpression. Examples of such disorders include, but are not limited to,conditions involving aberrant or deficient maturation or remodeling ofthe extracellular matrix, cellular proliferative and/or differentiativedisorders, neurological disorders, erythroid disorders, disordersassociated with skeletal integrity or bone metabolism, immune, e.g.inflammatory disorders, disorders associated with undesirable ordeficient vascularization/angiogenesis, disorders associated withundesirable extracellular matrix accumulation, e.g., characterized byfibrosis or a scar, liver disorders, or viral diseases.

[0019] The invention also provides assays for determining the activityof or the presence or absence of 53014 polypeptides or nucleic acidmolecules in a biological sample, including for disease diagnosis.

[0020] In a further aspect, the invention provides assays fordetermining the presence or absence of a genetic alteration in a 53014polypeptide or nucleic acid molecule, including for disease diagnosis.

[0021] In another aspect, the invention features a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence. At least one address of the pluralityhas a capture probe that recognizes a 53014 molecule. In one embodiment,the capture probe is a nucleic acid, e.g., a probe complementary to a53014 nucleic acid sequence. In another embodiment, the capture probe isa polypeptide, e.g., an antibody specific for 53014 polypeptides. Alsofeatured is a method of analyzing a sample by contacting the sample tothe aforementioned array and detecting binding of the sample to thearray.

[0022] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 depicts a hydropathy plot of human 53014. Relativelyhydrophobic residues are shown above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) are indicated by short vertical lines justbelow the hydropathy trace. The numbers corresponding to the amino acidsequence of human 53014 are indicated. Polypeptides of the inventioninclude fragments which include: all or part of a hydrophobic sequence,e.g., a sequence above the dashed line, e.g., the sequence from aboutamino acid 56 to 70, from about 155 to 168, and from about 300 to 315 ofSEQ ID NO: 2; all or part of a hydrophilic sequence, e.g., a sequencebelow the dashed line, e.g., the sequence from about amino acid 242 to253, from about 573 to 581, and from about 889 to 904 of SEQ ID NO: 2; asequence which includes a Cys, or a glycosylation site.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The full length human 53014 sequence (SEQ ID NO: 1), which isapproximately 5701 nucleotides long including untranslated regions,contains a predicted methionine-iniatiated coding sequence of about 3672nucleotides, including the termination codon (nucleotides indicated ascoding of SEQ ID NO: 1, SEQ ID NO: 3). The coding sequence encodes a1223 amino acid protein (SEQ ID NO: 2). The partial human 53014 sequence(SEQ ID NO: 4), which is approximately 5344 nucleotides long includinguntranslated regions, contains a predicted methionine-initiated codingsequence of about 3471 nucleotides, including the termination codon(nucleotides indicated as coding of SEQ ID NO: 4, SEQ ID NO: 6). Thecoding sequence of partial human 53014 encodes a 1156 amino acid protein(SEQ ID NO: 5).

[0025] The human 53014 protein of SEQ ID NO: 2 and FIG. 1 includes anamino-terminal hydrophobic amino acid sequence, consistent with a signalsequence, of about 22 amino acids (from amino acid 1 to about amino acid22 of SEQ ID NO: 2, PSORT, Nakai and Kanehisa (1992) Genomics14:897-911), which upon cleavage can result in the production of amature protein form. This mature protein form can be approximately 1201amino acid residues in length (from about amino acid 23 to amino acid1223 of SEQ ID NO: 2). In the event of acknowledgement of the signalpeptide in the 53014 sequence, the number “22” would be subtracted fromSEQ ID NO: 2 and the amino acid residue numbers for all motifs anddomains described herein would be renumbered accordingly. Amino acids120 to 240 of SEQ ID NO: 2 or amino acids 53 to 173 of SEQ ID NO: 5correspond to a “pro” region homologous to the reprolysin family(peptidase M12B family) propeptide (PFAM Accession Number PF01562),which is typically post-translationally cleaved upon conversion of thezymogen (inactive protein) to the catalytically active metalloprotease.Upon cleavage, the catalytically active mature protein can beapproximately 983 amino acid residues in length (from about amino acid241 to 1223 of SEQ ID NO: 2). In the event of acknowledgement of thepro-peptide in the 53014 sequence, the number “240” would be subtractedfrom SEQ ID NO: 2 (or “218” from a number for a sequence without thesignal peptide) or the number “173” would be subtracted from SEQ ID NO:5 and the amino acid residue numbers for all motifs and domainsdescribed herein for SEQ ID NOs: 2 and 5 would be renumberedaccordingly.

[0026] Human 53014 contains the following regions or other structuralfeatures (for general information regarding PFAM identifiers, PS prefixand PF prefix domain identification numbers, refer to Sonnhammer et al.(1997) Protein 28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html):

[0027] a reprolysin family propeptide domain (PFAM Accession NumberPF01562, SEQ ID NO: 7) located at about amino acid residues 120 to 240of SEQ ID NO: 2 or amino acids 53 to 173 of SEQ ID NO: 5;

[0028] a reprolysin domain (PFAM Accession Number PF01421, SEQ ID NO: 8)located at about amino acid residues 261 to 460 of SEQ ID NO: 2 or 194to 393 of SEQ ID NO: 5;

[0029] four thrombospondin type I domains (PFAM Accession NumberPF00090, SEQ ID NO: 9) at about amino acids 556 to 606, 849 to 906, 911to 968, and 970 to 1021 of SEQ ID NO: 2 or 489 to 539, 782 to 839, 844to 901, and 903 to 954 of SEQ ID NO: 5;

[0030] a signal peptide (PSORT, http://psort.nibb.ac jp.) located atabout amino acids 1 to 22 of SEQ ID NO: 2;

[0031] one leucine zipper pattern (Prosite PS00029, SEQ ID NO: 10)located at about amino acids 223 to 244 of SEQ ID NO: 2;

[0032] eleven protein kinase C phosphorylation sites (Prosite PS00005)located at about amino acids 32 to 34, 200 to 202, 247 to 249, 518 to520, 672 to 674, 696 to 698, 715 to 717, 774 to 776, 795 to 797, 943 to945, and 1029 to 1031 of SEQ ID NO: 2;

[0033] thirteen casein kinase II phosphorylation sites (Prosite PS00006)located at about amino acids 132 to 135, 169 to 172, 321 to 324, 348 to351, 360 to 363, 442 to 445, 477 to 480, 508 to 511, 609 to 612, 672 to675, 983 to 986, 1058 to 1061, and 1138 to 1141 of SEQ ID NO: 2;

[0034] two cAMP/cGMP-dependent protein kinase phosphorylation sites(Prosite PS00004) located at about amino acids 620 to 623 and 1095 to1098 of SEQ ID NO: 2;

[0035] four N-glycosylation sites (Prosite PS00001) located at aboutamino acids 109 to 112, 475 to 478, 941 to 944, and 1027 to 1030 of SEQID NO: 2;

[0036] one tyrosine kinase phosphorylation site (Prosite PS00007)located at about amino acids 543 to 549 of SEQ ID NO: 2; and

[0037] nineteen N-myristoylation sites (Prosite PS00008) located atabout amino acids 156 to 161, 163 to 168, 176 to 181, 301 to 306, 410 to415, 473 to 478, 550 to 555, 570 to 575, 587 to 592, 668 to 673, 695 to700, 704 to 709, 816 to 821, 866 to 871, 1023 to 1028, 1038 to 1043,1153 to 1158, 1174 to 1179, and 1186 to 1191 of SEQ ID NO: 2.

[0038] A plasmid containing the nucleotide sequence encoding human53014, named Fbh53014pat, was deposited with American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209,on ______ and assigned Accession Number ______. This deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. This deposit was made merely as a convenience for those ofskill in the art and is not an admission that a deposit is requiredunder 35 U.S.C. §112.

[0039] The 53014 protein contains a significant number of structuralcharacteristics in common with members of the metalloprotease family,e.g. the reprolysin (M12B) family of metalloproteases (e.g. the ADAMTSfamily). The term “family” when referring to the protein and nucleicacid molecules of the invention means two or more proteins or nucleicacid molecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin as well as other distinctproteins of human origin, or alternatively, can contain homologs ofnon-human origin, e.g., rat or mouse proteins. Members of a family alsocan have common functional characteristics.

[0040] As used herein, the term “metalloprotease” includes a protein orpolypeptide which is capable of binding a metal atom or ion and ofcatalyzing the cleavage of proteins, such as extracellular matrixproteins. This protein cleavage is a step in protein maturation,degradation of proteins, tumor growth, metastasis and angiogenesis,among others. A metalloprotease family, e.g. the reprolysin (M12B)family (e.g. the ADAMTS family) of proteins is characterized by a commonfold, a signal peptide, a propeptide, a zinc binding sequence, anddomains responsible for establishing the substrate identification,recognition or specificity or location of binding or action(localization) of the enzyme. The ADAMTS family of proteins is includedin the ADAMs family of reprolysin (M12B) proteins. The ADAMs familymembers are similar in domain organization, having from amino tocarboxyl termini, a signal peptide, a proregion, a zinc-metalloproteasecatalytic domain of the reprolysin type, a disintegrin domain, acysteine-rich domain, an epidermal growth factor-like domain, and inmany cases a membrane-spanning region and a cytoplasmic domain withsignaling potential (Wolfsberg et al. (1995) Developmental Biol.169:378-383; Wolfsberg et al. (1995) J. Cell Biol. 131:275-278;Hurskainen et al. (1999) J. Biol. Chem. 274:25555-25563). Members of theADAMs family of proteins include, but are not limited to, MDC (ADAM1),ADAMTS-1, fertilin (ADAM2), cryitestin (ADAM3), epididymal apicalprotein I, meltrin, MS2, TNF-a converting enzyme, Kuzbanian andmetargidin. ADAMTS-1 differs from other ADAM family members due to alack of the cysteine rich, EGF and transmembrane domains and theaddition of thrombospondin type I motifs (Kuno et al. (1997) J. Biol.Chem. 272: 556-562). The thrombospondin type I (TSP I) motifs inADAMTS-1 enable it to bind to the extracellular matrix (Kuno andMatsushima (1998) J. Biol. Chem. 273: 13912-13917). TSP I motifs areconserved domains in thrombospondin 1 and 2, multifunctional secretoryglycoproteins involved in the biological activities of blood clotting,neurite outgrowth, angiogenesis and regulating the proliferation,adhesion and migration of normal and tumor cells. The biologicalactivities of thrombospondin 1 and 2 are mediated by the binding of theTSP type I motifs to extracellular matrix molecules, such as heparansulfate, proteoglycans, fibronectin, laminin and collagen. The 53014polypeptide is homologous to a reprolysin (M12B) family member, humanprocollagen-1-N protease (SwissProt 095450). A GAP alignment of thisamino acid sequence to the 53014 polypeptide of SEQ ID NO: 2 found 50.8%identity between the two sequences (as calculated in matblas from theblosum50matrix). The 53014 polypeptide also is homologous to larynxcarcinoma-associated protein (larcap-1, GenBank accession numberAX211284). A GAP alignment of this amino acid sequence to the 53014polypeptide of SEQ ID NO: 2 found 76.6% overall identity, and 92.8%identity in the 1010 amino acid region of overlap between the twosequences (as calculated in matblas from the blosum50matrix).

[0041] A 53014 polypeptide is a predicted reprolysin (M12B) family (e.g.ADAMTS) family metalloprotease and can include a “reprolysin familypropeptide domain,” alternatively referred to as “peptidase M12Bpropeptide domain,” or regions homologous with a “reprolysin familypropeptide domain;” a “reprolysin domain”, alternatively referred to as“reprolysin (M12B) family zinc metalloprotease domain,” or a regionhomologous with a “reprolysin domain;” and at least one, two, three andpreferably four “thrombospondin domains,” or regions homologous with a“thrombospondin domain.”

[0042] As used herein, the term “reprolysin family propeptide domain,”or “propeptide domain” includes an amino acid sequence of about 50 to200 amino acid residues in length and having a bit score for thealignment of the sequence to the reprolysin family propeptide domain(HMM) of at least 30. Preferably a reprolysin family propeptide domainmediates maintenance of the metalloprotease in its inactive zymogenstate. Within this domain is a hydrophobic helical segment, from aboutamino acids 156 to 178 of SEQ ID NO: 2, which can participate instabilizing the quaternary association of the propeptide with thecatalytic domain in the zymogen state. Preferably, a propeptide domainincludes at least about 75 to 175 amino acid residues, more preferablyabout 100 to 150 amino acid residues of SEQ ID NO: 2, or about 110 to130 amino acids and has a bit score for the alignment of the sequence tothe reprolysin family propeptide domain (KMM) of at least 40, 45, 50 orgreater. The propeptide domain (HMM) has been assigned the PFAMAccession Numbers PF01562 (SEQ ID NO: 7,http;//genome.wustl.edu/Pfaml.html). An alignment of the reprolysinfamily propeptide domain (amino acids 120 to 240 of SEQ ID NO: 2 oramino acids 53 to 173 of SEQ ID NO: 5) of human 53014 with thisconsensus amino acid sequence derived from a hidden Markov model yieldeda bit score of 53.8.

[0043] In a preferred embodiment 53014 polypeptide or protein has a“reprolysin family propeptide domain” or “propeptide domain” or a regionwhich includes at least about 75 to 175, more preferably about 100 to150, most preferably 110 to 130 amino acid residues and has at leastabout 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a “reprolysinfamily propeptide domain,” e.g., the propeptide domain of human 53014(e.g., residues 120 to 240 of SEQ ID NO: 2 or 53 to 173 of SEQ ID NO:5).

[0044] As used herein, the term “reprolysin domain” or “reprolysin(M12B) family zinc metalloprotease domain” includes an amino acidsequence of about 150 to 320 amino acids residues in length and having abit score for the alignment of the sequence to the reprolysin domain(HMM) of at least 10. Preferably, a reprolysin domain includes at leastabout 165 to 275 amino acid residues, more preferably about 180 to 250amino acid residues, or about 190 to 210 amino acids and has a bit scorefor the alignment of the sequence to the protease domain (M) of at least15, 20, 25 or greater. Within this domain is a hydrophobic helicalsegment, from about amino acids 300 to 316 of SEQ ID NO: 2, which canparticipate in stabilizing the quaternary association of the catalyticdomain with the propeptide in the zymogen state. Preferably, thereprolysin domain has a metalloprotease activity, e.g., ametalloprotease activity as described herein. Preferably, the reprolysindomain includes a metal- (e.g., a zinc-) binding motif, which canparticipate in the metalloprotease activity. The reprolysin domain (HMM)has been assigned the PFAM Accession Numbers PF01421(http://genome.wustl.edu/Pfam/.html). An alignment of the reprolysindomain (amino acids 261 to 460 of SEQ ID NO: 2 or 194 to 393 of SEQ IDNO: 5) of human 53014 with a consensus amino acid sequence (SEQ ID NO:8) derived from a hidden Markov model yields a bit score of 25.5.

[0045] A reprolysin domain can include a Prosite neutral zincmetallopeptidases, zinc-binding signature sequence (PS00142, SEQ ID NO:11), or sequences homologous thereto to participate in themetalloprotease activity. This sequence has a consensus pattern:[GSTALIVN]-x(2)-H-E-[LIVMFYW]-{DEHRKP}-H-x-[LIVMFYWGSPQ]. The twoinvariant H residues bind zinc and the invariant E is an active siteresidue. In the above conserved signature sequence, and other motifs orsignature sequences described herein, the standard IUPAC one-letter codefor the amino acids is used. Each element in the pattern is separated bya dash (-); brackets ([]) indicate the particular residues that areaccepted at that position; braces ({}) indicate the residue in thatposition can be any amino acid except the residue(s) listed within; xindicates that any residue is accepted at that position; and numbers inparentheses (( )) indicate the number of residues represented by theaccompanying amino acid. A sequence similar to this consensus sequence,except for a conserved substitution of T after the E, can be found inthe 53014 polypeptide at about amino acids 395 to 404 of SEQ ID NO: 2,and thus the 53014 protein can participate in metalloprotease activity.

[0046] In a preferred embodiment 53014 polypeptide or protein has a“reprolysin domain” which includes at least about 165 to 275 amino acidresidues, more preferably about 180 to 250 amino acid residues, or about190 to 210 amino acid residues and has at least about 60%, 70%, 80%,90%, 95%, 99%, or 100% homology with a “reprolysin domain” of human53014 (e.g., residues 261 to 460 of SEQ ID NO: 2 or 194 to 393 of SEQ IDNO: 5).

[0047] As the 53014 metalloprotease has one or more thrombospondin (TSP)domains, a 53014 metalloprotease, subsequence or variant thereof canalso have a TSP domain or regions homologous with a TSP domain. A TSPdomain includes an amino acid sequence of about 15 to 95 amino acidresidues in length and having a bit score for the alignment of thesequence to the a reprolysin family propeptide domain (HMM) of at least5. Preferably, the thrombospondin domain has a TSP activity, e.g., a TSPactivity (e.g. mediate binding to other proteins such as extracellularmatrix molecules) as described herein. Preferably, a TSP domain includesat least about 30 to 80 amino acids, more preferably about 40 to 70amino acid residues, or about 50 to 60 amino acids and has a bit scorefor the alignment of the sequence to the TSP domain (HMM) of at least 8,10, 12 or greater. Alignments of the TSP domain (about amino acids 556to 606, 849 to 906, 911 to 968, and 970 to 1021 of SEQ ID NO: 2 or 489to 539, 782 to 839, 844 to 901, and 903 to 954 of SEQ ID NO: 5) of 53014metalloprotease with a consensus amino acid sequence derived from ahidden Markov model according to PFAM (SEQ ID NO: 9, PFAM AccessionNumbers PF00090) yield bit scores of 51.6, 13.4, 14.6, and 40.5,respectively.

[0048] In a preferred embodiment 53014 polypeptide or protein has atleast one thrombospondin domain or region which includes at least about30 to 80 amino acids, more preferably about 40 to 70 amino acidresidues, or about 50 to 60 amino acid residues and has at least about60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with a “thrombospondintype 1 domain,” e.g., the thrombospondin type 1 domain of human 53014(e.g., including residues 556 to 606, 849 to 906, 911 to 968, and 970 to1021 of SEQ ID NO: 2 or 489 to 539, 782 to 839, 844 to 901, and 903 to954 of SEQ ID NO: 5).

[0049] To identify the presence of a “reprolysin family propeptidedomain,” a “reprolysin” domain, or a “thrombospondin domain” in a 53014protein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the Pfam database of HMMs (e.g., thePfam database, release 2.1) using the default parameters(http://www.sanger.ac.uk/Software/Pfam HMM_search). For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28:405-420 and a detailed descriptionof HMMs can be found, for example, in Gribskov et al. (1990) Meth.Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; andStultz et al. (1993) Protein Sci. 2:305-314, the contents of which areincorporated herein by reference. A search was performed against the HMMdatabase resulting in the identification of a “reprolysin familypropeptide domain” at about residues 120 to 240 of SEQ ID NO: 2 or 53 to173 of SEQ ID NO: 5; a “reprolysin domain” in the amino acid sequence ofhuman 53014 at about residues 261 to 460 of SEQ ID NO: 2 or 194 to 393of SEQ ID NO: 5; and a “thrombospondin domain” at about residues 556 to606, 849 to 906, 911 to 968, or 970 to 1021 of SEQ ID NO: 2 or 489 to539, 782 to 839, 844 to 901, or 903 to 954 of SEQ ID NO: 5.

[0050] A human 53014 protein can further include a leucine zipper motif,a 53014 metalloprotease, subsequence or variant thereof can have aleucine zipper motif or regions homologous with a leucine zipper motif.Leucine zippers typically contain a repeat of leucine positioned everyseven amino acids (L-x(6)-L-x(6)-L-x(6)-L, SEQ ID NO: 10, PrositePS00029), over a distance of eight helical turns. The segmentscontaining these periodic arrays of leucines appear to exist in analpha-helical conformation in which leucine side chains extending fromone alpha-helix interact with those from a similar alpha helix of asecond polypeptide, facilitating dimerization. The leucine zipperpattern is present in many gene regulatory proteins, such as CCATT-boxand enhancer binding protein (C/EBP), cAMP response element (CRE)binding proteins (CREB, CRE-BP1, ATFs), jun/AP1 family transcriptionfactors, C-myc, L-myc and N-myc oncogenes and octamer-bindingtranscription factor 2 (Oct-2/OTF-2). These interactions are frequentlyrequired for the activity of the protein complex, e.g., transcriptionalactivation of a nucleic acid via binding to a gene regulatory sequenceand subsequent formation of a transcription initiation complex. Leucinezippers therefore mediate protein-protein interactions in vivo and inparticular, interactions between multi-subunit transcription factors(homodimers, heterodimers, etc.) Thus, in another embodiment, a 53014metalloprotease or subsequence or variant can have one or moreactivities of a leucine zipper motif, such as binding to anotherpolypeptide that has a leucine zipper, for example, forming a dimer witha 53014 metalloprotease or subsequence or variant containing a leucinezipper. The presence of a leucine zipper motif indicates that 53014metalloprotease can participate in different pathways due to an abilityto interact with different proteins via the leucine zipper motif. Forexample, the leucine zipper motif can allow 53014 metalloproteasebinding to a protein substrate which 53014 may then cleave. Thus, theleucine zipper motif modulates or is involved in one or more activitiesor functions of 53014 metalloprotease through its ability to conferbinding of 53014 metalloprotease to a target molecule or bindingpartner. The term “leucine zipper activity,” when used in reference to aprotein, means a protein having one or more activities associated withleucine-zipper function as described herein or otherwise known in theart. The leucine zipper motif can be found in the 53014 polypeptide atabout amino acids 223 to 244 of SEQ ID NO: 2.

[0051] A 53014 family member can include at least one reprolysin familypropeptide domain, at least one reprolysin domain, at least one, two,three, preferably four thrombospondin domains. A 53014 family memberalso can include at least one leucine zipper pattern. Furthermore, a53014 family member can include at least one, two, three, four, five,six, seven, eight, nine, ten, preferably eleven protein kinase Cphosphorylation sites (Prosite PS00005); at least one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve and preferablythirteen casein kinase II phosphorylation sites (Prosite PS00006); atleast one, two, three, preferably four N-glycosylation sites (PrositePS00001); at least one, preferably two cAMP/cGMP protein kinasephosphorylation sites (Prosite PS00004); at least one tyrosine kinasephosphorylation site (Prosite PS00007); and at least one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen, eighteen, and preferably nineteenN-myristoylation sites (Prosite PS00008).

[0052] As the 53014 polypeptides of the invention can modulate53014-mediated activities, they can be useful for developing noveldiagnostic and therapeutic agents for metalloprotease-associated orother 53014-associated disorders, as described below.

[0053] As used herein, a “metalloprotease-associated activity” includesan activity which involves binding a metal atom or ion, e.g. zinc,calcium, manganese or magnesium and catalyzing the cleavage of proteins,e.g. extracellular matrix proteins (e.g. collagen, fibronectin, gelatin,vitronectin, thrombospondin, aggrecan, fibrinogen, fibrin). Thisactivity can play a role in maturation or degradation of these proteins,tumor growth, metastasis, angiogenesis, neurite outgrowth, cellmigration, and tissue remodeling, among others. Thus, metalloproteasesare likely to play important roles in a wide range of diseasesincluding, but not limited to, cancer, neurological diseases, and avariety of inflammatory conditions. Examples of diseases in which ADAMTSproteins can play a role are Ehlers-Danlos syndrome type VIIC,characterized by the retention of the N-terminal propeptide of collagenI (Lenaers et al. (1971) Eur. J. Biochem. 23: 533-543) by ametalloprotease, procollagen I N-protease (Colige et al. (1999) Am. J.Hum. Genet. 65: 308-317); or cancer cachexia (Kuno et al. supra).

[0054] As used herein, a “53014 activity”, “biological activity of53014” or “functional activity of 53014”, refers to an activity exertedby a 53014 protein, polypeptide or nucleic acid molecule on e.g., a53014-responsive cell or on a 53014 substrate, e.g., a proteinsubstrate, as determined in vivo or in vitro. In one embodiment, a 53014activity is a direct activity, such as an association with a 53014target molecule. A “target molecule” or “binding partner” is a moleculewith which a 53014 protein binds or interacts in nature. In an exemplaryembodiment, 53014 is a metalloprotease, e.g. the reprolysin (M12B)family protease (e.g. an ADAMTS family member) and thus binds to orinteracts in nature with a metal atom or ion, e.g. zinc, calcium,manganese or magnesium and a macromolecule, e.g. an extracellular matrixmolecule or protein (e.g. collagen, fibronectin, gelatin, vitronectin,thrombospondin, aggrecan, fibrinogen, fibrin, heparan sulfate, dermatansulfate, chondroitin sulfate, bone sialoprotein, elastin or laminin).

[0055] A 53014 activity can also be an indirect activity, e.g., acellular signaling activity mediated by interaction of the 53014 proteinwith a 53014 receptor. Based on the above-described sequence structuresand similarities to molecules of known function, the 53014 molecules ofthe present invention can have similar biological activities asmetalloprotease family members. For example, the 53014 proteins of thepresent invention can have one or more of the following activities: (1)the ability to bind a metal atom or ion, e.g. zinc, calcium, manganeseor magnesium; (2) the ability to bind a macromolecule, e.g. anextracellular matrix molecule or protein (e.g. collagen, fibronectin,gelatin, vitronectin, thrombospondin, aggrecan, fibrinogen, fibrin,heparan sulfate, dermatan sulfate, chondroitin sulfate, bonesialoprotein, elastin or laminin); (3) the ability to modulate thepropeptide and convert from zymogen to active enzyme; (4) the ability tocleave a protein by hydrolysis of an amide bond in a protein; (5) theability to modulate cell signaling; (6) the ability to modulate cellproliferation; (7) the ability to modulate cell adhesion; (8) theability to modulate cell motility/migration; (9) the ability to modulateconnective tissue formation and maintenance; (10) the ability tomodulate an inflammatory response; or (11) the ability to modulateangiogenesis/vascularization.

[0056] The 53014 molecules of the invention can modulate the activitiesof cells in tissues where they are expressed. For example, high levelsof 53014 niRNA expression were found in brain cortex, spinal cord,hypothalamus, colon adenocarcinoma, lung tumor, ovary tumor, primaryosteoblasts, erythroid cells and the K562 erythroid cell line.Accordingly, the 53014 molecules of the invention can act as therapeuticor diagnostic agents for neurological, cellular proliferative and/ordifferentiative disorders, disorders associated with bone metabolism orerythroid disorders. Small amounts of 53014 mRNA expression also werefound in dorsal root ganglion, breast tumor, hemangioma, normal heart,chngestive heart failure heart, skeletal muscle, normal small intestine,normal breast, normal ovary, prostate adenocarcinoma, normal colon, andnormal tonsil. Trace amounts of 53014 mRNA expression were found incoronary smooth muscle cells, kidney, normal liver, normal femalebladder, normal adrenal gland, nerve, benign prostatic hypertrophyprostate, normal lung, inflammatory bowel disease colon, synovium,normal lymph node, normal spleen, macrophages, erythroid progenitors andactivated peripheral blood monocytes.

[0057] The 53014 molecules can be used to treat neurological disordersin part because the 53014 mRNA is expressed in the brain cortex, spinalcord and hypothalamus. Neurological disorders include disorders of thecentral nervous system (CNS) and the peripheral nervous system, e.g.,cognitive and neurodegenerative disorders. Examples of neurologicaldisorders include, but are not limited to, autonomic function disorderssuch as hypertension and sleep disorders, and neuropsychiatricdisorders, such as depression, schizophrenia, schizoaffective disorder,Korsakoff's psychosis, alcoholism, anxiety disorders, or phobicdisorders; learning or memory disorders, e.g., amnesia or age-relatedmemory loss, attention deficit disorder, dysthymic disorder, majordepressive disorder, mania, obsessive-compulsive disorder, psychoactivesubstance use disorders, anxiety, phobias, panic disorder, as well asbipolar affective disorder, e.g., severe bipolar affective (mood)disorder (BP-1), and bipolar affective neurological disorders, e.g.,migraine and obesity. Such neurological disorders include, for example,disorders involving neurons, and disorders involving glia, such asastrocytes, oligodendrocytes, ependymal cells, and microglia; cerebraledema, raised intracranial pressure and herniation, and hydrocephalus;malformations and developmental diseases, such as neural tube defects,forebrain anomalies, posterior fossa anomalies, and syringomyelia andhydromyelia; perinatal brain injury; cerebrovascular diseases, such asthose related to hypoxia, ischemia, and infarction, includinghypotension, hypoperfusion, and low-flow states—global cerebral ischemiaand focal cerebral ischemia—infarction from obstruction of local bloodsupply, intracranial hemorrhage, including intracerebral(intraparenchymal) hemorrhage, subarachnoid hemorrhage and rupturedberry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-borne (Arbo) viralencephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer's disease and Pick'sdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson's disease (paralysisagitans) and other Lewy diffuse body diseases, progressive supranuclearpalsy, corticobasal degenration, multiple system atrophy, includingstriatonigral degenration, Shy-Drager syndrome, and olivopontocerebellaratrophy, and Huntington's disease, senile dementia, Gilles de laTourette's syndrome, epilepsy, and Jakob-Creutzfieldt disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

[0058] The 53014 molecules can be used to treat cellular proliferativeand/or differentiative disorders in part because the 53014 mRNA isexpressed in the colon adenocarcinoma, lung tumor, ovary tumor, and isexpressed in only small or trace amounts in the corresponding normaltissue. Examples of cellular proliferative and/or differentiativedisorders include cancer, e.g., carcinoma, sarcoma, metastatic disordersor hematopoietic neoplastic disorders, e.g., leukemias. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.Examples of tissues in which the 53014 molecules of the invention can beused to treat cellular proliferative and/or differentiative disordersare colon, lung and ovary.

[0059] As used herein, the term “cancer” (also used interchangeably withthe terms, “hyperproliferative” and “neoplastic”) refers to cells havingthe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Cancerous diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, e.g., malignant tumor growth,characterizing or constituting a cancer-associated condition, e.g.,cachexia, or may be categorized as non-pathologic, i.e., a deviationfrom normal but not associated with a disease state, e.g., cellproliferation associated with wound repair. The term is meant to includeall types of cancerous growths or oncogenic processes, metastatictissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type or stage of invasiveness. The term“cancer” includes malignancies of the various organ systems, such asthose affecting lung, breast, thyroid, lymphoid, gastrointestinal, andgenito-urinary tract, as well as adenocarcinomas which includemalignancies such as most colon cancers, renal-cell carcinoma, prostatecancer and/or testicular tumors, non-small cell carcinoma of the lung,cancer of the small intestine and cancer of the esophagus. The term“carcinoma” is art recognized and refers to malignancies of epithelialor endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term “carcinoma” also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures. The term “sarcoma” is art recognizedand refers to malignant tumors of mesenchymal derivation.

[0060] The 53014 molecules of the invention can be used to monitor,treat and/or diagnose a variety of proliferative disorders. Suchdisorders include hematopoietic neoplastic disorders. As used herein,the term “hematopoietic neoplastic disorders” includes diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Preferably, the diseases arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional exemplary myeloid disorders include, but are notlimited to, acute promyeloid leukemia (APML), acute myelogenous leukemia(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus (1991)Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include,but are not limited to acute lymphoblastic leukemia (ALL) which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

[0061] The 53014 molecules can be used to treat disorders associatedwith skeletal integrity and bone metabolism in part because the 53014mRNA is expressed in primary osteoblasts. “Skeletal integrity” refers todirect or indirect effects on the formation or maintenance of bones orjoints and the tissues, such as ligaments, tendons or muscles whichconnect and control movement of those structures. “Bone metabolism”refers to direct or indirect effects in the formation or degeneration ofbone structures, e.g., bone formation, bone resorption, etc., which canultimately affect the concentrations in serum of calcium and phosphate.This term also includes activities mediated by 53014 molecules in bonecells, e.g. osteoclasts and osteoblasts, that can in turn result in boneformation and degeneration. For example, 53014 molecules can supportdifferent activities of bone resorbing osteoclasts such as thestimulation of differentiation of monocytes and mononuclear phagocytesinto osteoclasts. Accordingly, 53014 molecules that modulate theproduction of bone cells can influence bone formation and degeneration,and thus can be used to treat bone disorders. Examples of such disordersinclude, but are not limited to, osteogenesis imperfecta, osteoporosis,osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica,Ehlers-Danlos syndrome, renal osteodystrophy, osteosclerosis,anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium,secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism,cirrhosis, obstructive jaundice, drug induced metabolism, medullarycarcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoidantagonism, malabsorption syndrome, steatorrhea, tropical sprue,idiopathic hypercalcemia and milk fever.

[0062] The 53014 molecules can be used to treat erythroid disorders inpart because the 53014 mRNA is expressed in the erythroid cells and theK562 erythroid cell line.

[0063] As used herein, the term “erythroid associated disorders” includedisorders involving aberrant (increased or deficient) erythroblastproliferation, e.g., an erythroleukemia, and aberrant (increased ordeficient) erythroblast differentiation, e.g., an anemia.Erythrocyte-associated disorders include anemias such as, for example,drug-(chemotherapy-) induced anemias, hemolytic anemias due tohereditary cell membrane abnormalities, such as hereditaryspherocytosis, hereditary elliptocytosis, and hereditarypyropoikilocytosis; hemolytic anemias due to acquired cell membranedefects, such as paroxysmal nocturnal hemoglobinuria and spur cellanemia; hemolytic anemias caused by antibody reactions, for example tothe RBC antigens, or antigens of the ABO system, Lewis system, Iisystem, Rh system, Kidd system, Duffy system, and Kell system;methemoglobinemia; a failure of erythropoiesis, for example, as a resultof aplastic anemia, pure red cell aplasia, myelodysplastic syndromes,sideroblastic anemias, and congenital dyserythropoietic anemia;secondary anemia in non-hematolic disorders, for example, as a result ofchemotherapy, alcoholism, or liver disease; anemia of chronic disease,such as chronic renal failure; and endocrine deficiency diseases.

[0064] Agents that modulate 53014 polypeptide or nucleic acid activityor expression can be used to treat anemias, in particular, drug-inducedanemias or anemias associated with cancer chemotherapy, chronic renalfailure, malignancies, adult and juvenile rheumatoid arthritis,disorders of hemoglobin synthesis, prematurity, and zidovudine treatmentof HIV infection. A subject receiving the treatment can be additionallytreated with a second agent, e.g., erythropoietin, to further amelioratethe condition.

[0065] As used herein, the term “erythropoietin” or “EPO” refers to aglycoprotein produced in the kidney, which is the principal hormoneresponsible for stimulating red blood cell production (erythrogenesis).EPO stimulates the division and differentiation of committed erythroidprogenitors in the bone marrow. Normal plasma erythropoietin levelsrange from 0.01 to 0.03 Units/mL, and can increase up to 100 to1,000-fold during hypoxia or anemia. Graber and Krantz (1978) Ann. Rev.Med. 29:51; Eschbach and Adamson (1985) Kidney Intl. 28:1. Recombinanthuman erythropoietin (rHuEpo or epoietin alpha) is commerciallyavailable as EPOGEN.RTM. (epoietin alpha, recombinant humanerythropoietin) (Amgen Inc., Thousand Oaks, Calif.) and as PROCRIT.RTM.(epoietin alpha, recombinant human erythropoietin) (Ortho Biotech Inc.,Raritan, N.J.).

[0066] Another example of an erythroid-associated disorder iserythrocytosis. Erythrocytosis, a disorder of red blood celloverproduction caused by excessive and/or ectopic erythropoietinproduction, can be caused by cancers, e.g., a renal cell cancer, ahepatocarcinoma, and a central nervous system cancer. Diseasesassociated with erythrocytosis include polycythemias, e.g., polycythemiavera, secondary polycythemia, and relative polycythemia.

[0067] Thus, the 53014 molecules can act as novel diagnostic targets andtherapeutic agents for controlling one or more neurological, cellularproliferative and/or differentiative disorders, disorders associatedwith bone metabolism or erythroid disorders or other metalloproteasedisorders. As used herein, “metalloprotease disorders” are diseases ordisorders whose pathogenesis is caused by, is related to, or isassociated with aberrant or deficient metalloprotease protein functionor expression. Metalloprotease associated disorders can detrimentallyaffect cell proliferation, cell adhesion, cell motility and migration,tissue structural integrity (e.g., connective tissue formation andmaintenance), inflammatory response, erythroid cell activity, geneexpression; or angiogenesis and vascularization, among others. Thus,examples of metalloprotease associated disorders in which the 53014molecules of the invention can be directly or indirectly involvedinclude cellular proliferative and/or differentiative disorders;disorders associated with undesirable or deficientvascularization/angiogenesis; disorders associated with undesirable ordeficient cell adhesion, motility or migration, including, e.g.,metastasis; disorders associated with undesirable or deficient tissuestructural integrity; disorders associated with undesirableextracellular matrix accumulation, e.g., characterized by fibrosis or ascar; inflammatory disorders, erythroid cell associated disorders; geneexpression disorders; and bleeding/clotting disorders. Examples of suchdisorders, e.g., metalloprotease-associated or other 53014-associateddisorders, include but are not limited to, cellular proliferative and/ordifferentiative disorders, erythroid cell associated disorders, asdescribed above; immune e.g., inflammatory, disorders, disordersassociated with undesirable or deficient vascularization/angiogenesis,disorders associated with undesirable extracellular matrix accumulation,e.g., characterized by fibrosis or a scar, liver disorders, or viraldiseases.

[0068] The 53014 nucleic acid and protein of the invention can be usedto treat and/or diagnose a variety of immune, e.g., inflammatory, (e.g.respiratory inflammatory) disorders in part because metalloproteasefamily members are involved in inflammatory activities. Examples ofimmune disorders or diseases include, but are not limited to, autoimmunediseases (including, for example, diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, inflammatorybowel disease, e.g. Crohn's disease and ulcerative colitis, aphthousulcer, iritis, conjunctivitis, keratoconjunctivitis, asthma, allergicasthma, chronic obstructive pulmonary disease, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitisposterior, and interstitial lung fibrosis), graft-versus-host disease,cases of transplantation, and allergy such as, atopic allergy.

[0069] The 53014 molecules can be used to treat disorders associatedwith undesirable or deficient vascularization/angiogenesis disorders inpart because metalloprotease family members are found in angiogenicactivities. As used herein, an “angiogenic disorder” includes a diseaseor disorder which affects or is caused by aberrant or deficientangiogenesis. Disorders involving angiogenesis include, but are notlimited to, aberrant or excess angiogenesis in tumors such ashemangiomas and Kaposi's sarcoma, von Hippel-Lindau disease, as well asthe angiogenesis associated with tumor growth; aberrant or excessangiogenesis in diseases such as a Castleman's disease or fibrodysplasiaossificans progressiva; aberrant or deficient angiogenesis associatedwith aging, complications of healing certain wounds and complications ofdiseases such as diabetes and rheumatoid arthritis; or aberrant ordeficient angiogenesis associated with hereditary hemorrhagictelangiectasia, autosomal dominant polycystic kidney disease,myelodysplastic syndrome or Klippel-Trenaunay-Weber syndrome.

[0070] The 53014 molecules can be used to treat or diagnose hepaticdisorders in part because aberrant or deficient function or expressionof metalloprotease family members can result in disorders of the liver.Examples of such disorders include, but are not limited to, disordersassociated with an accumulation in the liver of fibrous tissue, such asthat resulting from an imbalance between production and degradation ofthe extracellular matrix accompanied by the collapse and condensation ofpreexisting fibers. The methods described herein can be used to diagnoseor treat hepatocellular necrosis or injury induced by a wide variety ofagents including processes which disturb homeostasis, such as aninflammatory process, tissue damage resulting from toxic injury oraltered hepatic blood flow, and infections (e.g., bacterial, viral andparasitic). For example, the methods can be used for the early detectionof hepatic injury, such as portal hypertension or hepatic fibrosis. Inaddition, the methods can be employed to detect liver fibrosisattributed to inborn errors of metabolism, for example, fibrosisresulting from a storage disorder such as Gaucher's disease (lipidabnormalities) or a glycogen storage disease, Al-antitrypsin deficiency;a disorder mediating the accumulation (e.g., storage) of an exogenoussubstance, for example, hemochromatosis (iron-overload syndrome) andcopper storage diseases (Wilson's disease), disorders resulting in theaccumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia andgalactosemia) and peroxisomal disorders (e.g., Zellweger syndrome).Additionally, the methods described herein can be used for the earlydetection and treatment of liver injury associated with theadministration of various chemicals or drugs, such as for example,methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine,tolbutamide or alcohol, or which represents a hepatic manifestation of avascular disorder such as obstruction of either the intrahepatic orextrahepatic bile flow or an alteration in hepatic circulationresulting, for example, from chronic heart failure, veno-occlusivedisease, portal vein thrombosis or Budd-Chiari syndrome.

[0071] Additionally, 53014 molecules can play an important role in theetiology of certain viral diseases, including but not limited toHepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of53014 activity could be used to control viral diseases. The modulatorscan be used in the treatment and/or diagnosis of viral infected tissueor virus-associated tissue fibrosis, especially liver and liverfibrosis. Also, 53014 modulators can be used in the treatment and/ordiagnosis of virus-associated carcinoma, especially hepatocellularcancer.

[0072] The 53014 protein, fragments thereof, and derivatives and othervariants of the sequence in SEQ ID NO: 2 or SEQ ID NO: 5 thereof arecollectively referred to as “polypeptides or proteins of the invention”or “53014 polypeptides or proteins”. Nucleic acid molecules encodingsuch polypeptides or proteins are collectively referred to as “nucleicacids of the invention” or “53014 nucleic acids.”

[0073] As used herein, the term “nucleic acid molecule” includes DNAmolecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., anmRNA) and analogs of the DNA or RNA generated, e.g., by the use ofnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

[0074] The term “isolated or purified nucleic acid molecule” includesnucleic acid molecules which are separated from other nucleic acidmolecules which are present in the natural source of the nucleic acid.For example, with regards to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

[0075] As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

[0076] As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein).

[0077] As used herein, the terms “gene” and “recombinant gene” refer tonucleic acid molecules which include an open reading frame encoding a53014 protein, preferably a mammalian 53014 protein, and can furtherinclude non-coding regulatory sequences, and introns.

[0078] An “isolated” or “purified” polypeptide or protein issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the protein is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. In one embodiment, the language “substantiallyfree” means preparation of 53014 protein having less than about 30%,20%, 10% and more preferably 5% (by dry weight), of non-53014 protein(also referred to herein as a “contaminating protein”), or of chemicalprecursors or non-53014 chemicals. When the 53014 protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The invention includes isolated or purified preparations ofat least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0079] A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of 53014 (e.g., the sequence of SEQID NO: 1, 3, 4 or 6) without abolishing or more preferably, withoutsubstantially altering a biological activity, whereas an “essential”amino acid residue results in such a change. For example, amino acidresidues that are conserved among the polypeptides of the presentinvention, e.g., those present in the reprolysin family propeptidedomain, the reprolysin domain, or one, two, three or all fourthrombospondin domains are predicted to be particularly unamenable toalteration.

[0080] A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a 53014protein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a 53014 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for 53014 biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4 or SEQ ID NO: 6, the encoded protein can be expressedrecombinantly and the activity of the protein can be determined.

[0081] As used herein, a “biologically active portion” of a 53014protein includes a fragment of a 53014 protein which participates in aninteraction between a 53014 molecule and a non-53014 molecule.Biologically active portions of a 53014 protein include peptidescomprising amino acid sequences sufficiently homologous to or derivedfrom the amino acid sequence of the 53014 protein, e.g., the amino acidsequence shown in SEQ ID NO: 2, or SEQ ID NO: 5 which include feweramino acids than the full length 53014 protein, and exhibit at least oneactivity of a 53014 protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the 53014protein, e.g., the ability to bind a metal atom or ion, e.g. zinc,calcium, manganese or magnesium or the ability to bind or catalyze thecleavage of a macromolecule, e.g. an extracellular matrix molecule orprotein (e.g. collagen, fibronectin, gelatin, vitronectin,thrombospondin, aggrecan, fibrinogen, fibrin, heparan sulfate, dermatansulfate, chondroitin sulfate, bone sialoprotein, elastin or laminin). Abiologically active portion of a 53014 protein can be a polypeptidewhich is, for example, 10, 25, 50, 100, 200 or more amino acids inlength. Biologically active portions of a 53014 protein can be used astargets for developing agents which modulate a 53014 mediated activity,e.g., the ability to bind a metal atom or ion, e.g. zinc, calcium,manganese or magnesium or the ability to bind or catalyze the cleavageof a macromolecule, e.g. an extracellular matrix molecule or protein(e.g. collagen, fibronectin, gelatin, vitronectin, thrombospondin,aggrecan, fibrinogen, fibrin, heparan sulfate, dermatan sulfate,chondroitin sulfate, bone sialoprotein, elastin or laminin).

[0082] Calculations of homology or sequence identity (the terms“homology” and “identity” are used interchangeably herein) betweensequences are performed as follows:

[0083] To determine the percent identity of two amino acid sequences, orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, even more preferably at least 60%,and even more preferably at least 70%, 80%, 90%, 100% of the length ofthe reference sequence (e.g., when aligning a second sequence to the53014 amino acid sequence of SEQ ID NO: 2 having 1223 amino acidresidues, at least [30%] 366, preferably at least [40%] 489, morepreferably at least [50%] 611, even more preferably at least [60%] 733,and even more preferably at least [70%] 856, [80%] 978, or [90%] 1100amino acid residues are aligned). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0084] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (1970)J. Mol. Biol. 48:444-453 algorithm which has been incorporated into theGAP program in the GCG software package (available athttp:/Hwww.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) are a Blossum 62 scoring matrixwith a gap penalty of 12, a gap extend penalty of 4, and a frameshiftgap penalty of 5.

[0085] The percent identity between two amino acid or nucleotidesequences can be determined using the algorithm of Meyers and Miller((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

[0086] The nucleic acid and protein sequences described herein can beused as a “query sequence” to perform a search against public databasesto, for example, identify other family members or related sequences.Such searches can be performed using the NBLAST and XBLAST programs(version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 53014 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 53014 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

[0087] Particular 53014 polypeptides of the present invention have anamino acid sequence substantially identical to the amino acid sequenceof SEQ ID NO: 2 or SEQ ID NO: 5. In the context of an amino acidsequence, the term “substantially identical” is used herein to refer toa first amino acid that contains a sufficient or minimum number of aminoacid residues that are i) identical to, or ii) conservativesubstitutions of aligned amino acid residues in a second amino acidsequence such that the first and second amino acid sequences can have acommon structural domain and/or common functional activity. For example,amino acid sequences that contain a common structural domain having atleast about 60%, or 65% identity, likely 75% identity, more likely 85%,90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ IDNO: 2 or SEQ ID NO: 5 are termed substantially identical.

[0088] In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 60%, or 65%identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 3, 4, or 6 aretermed substantially identical.

[0089] “Misexpression or aberrant expression”, as used herein, refers toa non-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

[0090] “Subject”, as used herein, can refer to a mammal, e.g., a human,or to an experimental or animal or disease model. The subject can alsobe a non-human animal, e.g., a horse, cow, goat, or other domesticanimal.

[0091] A “purified preparation of cells”, as used herein, refers to, inthe case of plant or animal cells, an in vitro preparation of cells andnot an entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

[0092] Various aspects of the invention are described in further detailbelow.

[0093] Isolated Nucleic Acid Molecules

[0094] In one aspect, the invention provides, an isolated or purified,nucleic acid molecule that encodes a 53014 polypeptide described herein,e.g., a full length 53014 protein or a fragment thereof, e.g., abiologically active portion of 53014 protein. Also included is a nucleicacid fragment suitable for use as a hybridization probe, which can beused, e.g., to identify a nucleic acid molecule encoding a polypeptideof the invention, 53014 mRNA, and fragments suitable for use as primers,e.g., PCR primers for the amplification or mutation of nucleic acidmolecules.

[0095] In one embodiment, an isolated nucleic acid molecule of theinvention includes the nucleotide sequence shown in SEQ ID NO: 1, or aportion of any of this nucleotide sequence. In one embodiment, thenucleic acid molecule includes sequences encoding the human 53014protein (i.e., “the coding region” of SEQ ID NO: 1, as shown in SEQ IDNO: 3), as well as 5′ untranslated sequences (nucleotides 1 to 372 ofSEQ ID NO: 1) and 3′ untranslated sequences (nucleotides 4045 to 5701 ofSEQ ID NO: 1). Alternatively, the nucleic acid molecule can include onlythe coding region of SEQ ID NO: 1 (e.g., SEQ ID NO: 3) and, e.g., noflanking sequences which normally accompany the subject sequence. Inanother embodiment, the nucleic acid molecule encodes a sequencecorresponding to a fragment of the protein from about amino acid 261 to460 of SEQ ID NO: 2, or a fragment thereof, e.g. about amino acidresidues 261 to 325, 326 to 400, or 401 to 460 of SEQ ID NO: 2.

[0096] In one embodiment, an isolated nucleic acid molecule of theinvention includes the nucleotide sequence shown in SEQ ID NO: 4, or aportion of any of these nucleotide sequences. In one embodiment, thenucleic acid molecule includes sequences encoding the human 53014protein (i.e., “the coding region” of SEQ ID NO: 4, as shown in SEQ IDNO: 6), as well as 5′ untranslated sequences (about nucleotides 1 to 216of SEQ ID NO: 4) and 3′ untranslated sequences (about nucleotides 3687to 5343 of SEQ ID NO: 4). Alternatively, the nucleic acid molecule caninclude only the coding region of SEQ ID NO: 4 (e.g., SEQ ID NO: 6) and,e.g., no flanking sequences which normally accompany the subjectsequence. In another embodiment, the nucleic acid molecule encodes asequence corresponding to a fragment of the protein from about aminoacid 53 to 173, 194 to 393, 489 to 539, 782 to 839, 844 to 901, or 903to 954 of SEQ ID NO: 5.

[0097] In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or a portionof any of these nucleotide sequences. In other embodiments, the nucleicacid molecule of the invention is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4,or SEQ ID NO: 6 such that it can hybridize to the nucleotide sequenceshown in SEQ ID NO: 1, 3, 4, or 6, thereby forming a stable duplex.

[0098] In one embodiment, an isolated nucleic acid molecule of thepresent invention includes a nucleotide sequence which is at leastabout: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more homologous to the entire length of the nucleotidesequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:6, or a portion, preferably of the same length, of any of thesenucleotide sequences.

[0099] 53014 Nucleic Acid Fragments

[0100] A nucleic acid molecule of the invention can include only aportion of the nucleic acid sequence of SEQ ID NO: 1, 3, 4, or 6. Forexample, such a nucleic acid molecule can include a fragment which canbe used as a probe or primer or a fragment encoding a portion of a 53014protein, e.g., an immunogenic or biologically active portion of a 53014protein. A fragment can comprise those nucleotides of SEQ ID NO: 1 orSEQ ID NO: 4, which encode the reprolysin family propeptide domain, thereprolysin domain, or a thrombospondin domain of human 53014. Thenucleotide sequence determined from the cloning of the 53014 gene allowsfor the generation of probes and primers designed for use in identifyingand/or cloning other 53014 family members, or fragments thereof, as wellas 53014 homologs, or fragments thereof, from other species.

[0101] In another embodiment, a nucleic acid includes a nucleotidesequence that includes part, or all, of the coding region and extendsinto either (or both) the 5′ or 3′ noncoding region. Other embodimentsinclude a fragment which includes a nucleotide sequence encoding anamino acid fragment described herein. Nucleic acid fragments can encodea specific domain or site described herein or fragments thereof,particularly fragments thereof which are at least 150 amino acids inlength. Fragments also include nucleic acid sequences corresponding tospecific amino acid sequences described above or fragments thereof.Nucleic acid fragments should not to be construed as encompassing thosefragments that may have been disclosed prior to the invention.

[0102] A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, a 53014 nucleic acid fragment caninclude a sequence corresponding to a reprolysin family propeptidedomain, a reprolysin domain, or a thrombospondin domain, as describedherein.

[0103] 53014 probes and primers are provided. Typically a probe/primeris an isolated or purified oligonucleotide. The oligonucleotidetypically includes a region of nucleotide sequence that hybridizes understringent conditions to at least about 7, 12 or 15, preferably about 20or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense or antisense sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6, or of a naturallyoccurring allelic variant or mutant of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 4, or SEQ ID NO: 6.

[0104] In a preferred embodiment the nucleic acid is a probe which is atleast 5 or 10, and less than 200, more preferably less than 100, or lessthan 50, base pairs in length. It should be identical, or differ by 1,or less than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

[0105] A probe or primer can be derived from the sense or anti-sensestrand of a nucleic acid which encodes: a reprolysin family propeptidedomain from about amino acid residues 120 to 240 of SEQ ID NO: 2; or 53to 173 of SEQ ID NO: 5, the reprolysin domain from about amino acidresidues 261 to 460 of SEQ ID NO: 2 or 194 to 393 of SEQ ID NO: 5, or athrombospondin domain from about amino acid residues 556 to 606, 849 to906, 911 to 968, or 970 to 1021 of SEQ ID NO: 2 or 489 to 539, 782 to839, 844 to 901, or 903 to 954 of SEQ ID NO: 5.

[0106] In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a 53014 sequence, e.g., a domain, region, site or othersequence described herein. The primers should be at least 5, 10, or 50base pairs in length and less than 100, or less than 200, base pairs inlength. The primers should be identical, or differ by one base from asequence disclosed herein or from a naturally occurring variant. Forexample, primers suitable for amplifying all or a portion of any of thefollowing regions are provided: a reprolysin family propeptide domainfrom about amino acid residues 120 to 240 of SEQ ID NO: 2; or 53 to 173of SEQ ID NO: 5, the reprolysin domain from about amino acid residues261 to 460 of SEQ ID NO: 2 or 194 to 393 of SEQ ID NO: 5, or athrombospondin domain from about amino acid residues 556 to 606, 849 to906, 911 to 968, or 970 to 1021 of SEQ ID NO: 2 or 489 to 539, 782 to839, 844 to 901, or 903 to 954 of SEQ ID NO: 5.

[0107] A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

[0108] A nucleic acid fragment encoding a “biologically active portionof a 53014 polypeptide” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO: 1 or 3, which encodes a polypeptidehaving a 53014 biological activity (e.g., the biological activities ofthe 53014 proteins are described herein), expressing the encoded portionof the 53014 protein (e.g., by recombinant expression in vitro) andassessing the activity of the encoded portion of the 53014 protein. Forexample, a nucleic acid fragment encoding a biologically active portionof 53014 includes a reprolysin family propeptide domain from about aminoacid residues 120 to 240 of SEQ ID NO: 2; or 53 to 173 of SEQ ID NO: 5,the reprolysin domain from about amino acid residues 261 to 460 of SEQID NO: 2 or 194 to 393 of SEQ ID NO: 5, or a thrombospondin domain fromabout amino acid residues 556 to 606, 849 to 906, 911 to 968, or 970 to1021 of SEQ ID NO: 2 or 489 to 539, 782 to 839, 844 to 901, or 903 to954 of SEQ ID NO: 5. A nucleic acid fragment encoding a biologicallyactive portion of a 53014 polypeptide, can comprise a nucleotidesequence which is greater than 450 or more nucleotides in length.

[0109] In preferred embodiments, a nucleic acid includes a nucleotidesequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300,2400, 2500, 2600, 2600, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500,3600, 3700, 3800, 3900,4000, 4100, 4200, 4300, 4400,4500, 4600, 4700,4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 4, or SEQ ID NO: 6.

[0110] 53014 Nucleic Acid Variants

[0111] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, or SEQ ID NO: 6. Such differences can be due to degeneracyof the genetic code (and result in a nucleic acid which encodes the same53014 proteins as those encoded by the nucleotide sequence disclosedherein. In another embodiment, an isolated nucleic acid molecule of theinvention has a nucleotide sequence encoding a protein having an aminoacid sequence which differs, by at least 1, but less than 5, 10, 20, 50,or 100 amino acid residues that shown in SEQ ID NO: 2 or SEQ ID NO: 5.If alignment is needed for this comparison the sequences should bealigned for maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

[0112] Nucleic acids of the inventor can be chosen for having codons,which are preferred, or non-preferred, for a particular expressionsystem. E.g., the nucleic acid can be one in which at least one codon,at preferably at least 10%, or 20% of the codons has been altered suchthat the sequence is optimized for expression in E. coli, yeast, human,insect, or CHO cells.

[0113] Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

[0114] In a preferred embodiment, the nucleic acid differs from that ofSEQ ID NO: 1, 3, 4 or 6, e.g., as follows: by at least one but less than10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or20% of the nucleotides in the subject nucleic acid. If necessary forthis analysis the sequences should be aligned for maximum homology.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.

[0115] Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the nucleotidesequence shown in SEQ ID NO: 2, SEQ ID NO: 5, or a fragment of thissequence. Such nucleic acid molecules can readily be identified as beingable to hybridize under stringent conditions, to the nucleotide sequenceshown in SEQ ID NO 2, SEQ ID NO: 5, or a fragment of the sequence.Nucleic acid molecules corresponding to orthologs, homologs, and allelicvariants of the 53014 cDNAs of the invention can further be isolated bymapping to the same chromosome or locus as the 53014 gene.

[0116] Preferred variants include those that are correlated with bindinga metal atom or ion, e.g. zinc, calcium, manganese or magnesium orbinding or catalyzing the cleavage of a macromolecule, e.g. anextracellular matrix molecule or protein (e.g. collagen, fibronectin,gelatin, vitronectin, thrombospondin, aggrecan, fibrinogen, fibrin,heparan sulfate, dermatan sulfate, chondroitin sulfate, bonesialoprotein, elastin or laminin).

[0117] Allelic variants of 53014, e.g., human 53014, include bothfunctional and non-functional proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the 53014 proteinwithin a population that maintain the ability to bind a metal atom orion, e.g. zinc, calcium, manganese or magnesium or the ability to bindor catalyze the cleavage of a macromolecule, e.g. an extracellularmatrix molecule or protein (e.g. collagen, fibronectin, gelatin,vitronectin, thrombospondin, aggrecan, fibrinogen, fibrin, heparansulfate, dermatan sulfate, chondroitin sulfate, bone sialoprotein,elastin or laminin). Functional allelic variants will typically containonly conservative substitution of one or more amino acids of SEQ ID NO:2, SEQ ID NO: 5, or substitution, deletion or insertion of non-criticalresidues in non-critical regions of the protein. Non-functional allelicvariants are naturally-occurring amino acid sequence variants of the53014, e.g., human 53014, protein within a population that do not havethe ability to bind a metal atom or ion, e.g. zinc, calcium, manganeseor magnesium or bind or catalyze the cleavage of a macromolecule, e.g.an extracellular matrix molecule or protein (e.g. collagen, fibronectin,gelatin, vitronectin, thrombospondin, aggrecan, fibrinogen, fibrin,heparan sulfate, dermatan sulfate, chondroitin sulfate, bonesialoprotein, elastin or laminin). Non-functional allelic variants willtypically contain a non-conservative substitution, a deletion, orinsertion, or premature truncation of the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 5, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

[0118] Moreover, nucleic acid molecules encoding other 53014 familymembers and, thus, which have a nucleotide sequence which differs fromthe 53014 sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQID NO: 6 are intended to be within the scope of the invention.

[0119] Antisense Nucleic Acid Molecules, Ribozymes and Modified 53014Nucleic Acid Molecules

[0120] In another aspect, the invention features, an isolated nucleicacid molecule which is antisense to 53014. An “antisense” nucleic acidcan include a nucleotide sequence which is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. The antisense nucleic acid can be complementary to an entire53014 coding strand, or to only a portion thereof (e.g., the codingregion of human 53014 corresponding to SEQ ID NO: 3 or SEQ ID NO: 6). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding 53014 (e.g., the 5′ and 3′ untranslated regions).

[0121] An antisense nucleic acid can be designed such that it iscomplementary to the entire coding region of 53014 mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of 53014 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of 53014 mRNA, e.g., between the −10 and +10regions of the target gene nucleotide sequence of interest. An antisenseoligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0122] An antisense nucleic acid of the invention can be constructedusing chemical synthesis and enzymatic ligation reactions usingprocedures known in the art. For example, an antisense nucleic acid(e.g., an antisense oligonucleotide) can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. The antisense nucleicacid also can be produced biologically using an expression vector intowhich a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest, describedfurther in the following subsection).

[0123] The antisense nucleic acid molecules of the invention aretypically administered to a subject (e.g., by direct injection at atissue site), or generated in situ such that they hybridize with or bindto cellular mRNA and/or genomic DNA encoding a 53014 protein to therebyinhibit expression of the protein, e.g., by inhibiting transcriptionand/or translation. Alternatively, antisense nucleic acid molecules canbe modified to target selected cells and then administered systemically.For systemic administration, antisense molecules can be modified suchthat they specifically or selectively bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0124] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an ox-anomeric nucleic acid molecule. An a-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0125] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. A ribozyme having specificity for a53014-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a 53014 cDNA disclosedherein (i.e., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:6), and a sequence having known catalytic sequence responsible for mRNAcleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988)Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the nucleotide sequence of the activesite is complementary to the nucleotide sequence to be cleaved in a53014-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; andCech et al. U.S. Pat. No. 5,116,742. Alternatively, 53014 mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

[0126] 53014 gene expression can be inhibited by targeting nucleotidesequences complementary to the regulatory region of the 53014 (e.g., the53014 promoter and/or enhancers) to form triple helical structures thatprevent transcription of the 53014 gene in target cells. See generally,Helene (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N.Y.Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15. Thepotential sequences that can be targeted for triple helix formation canbe increased by creating a so-called “switchback” nucleic acid molecule.Switchback molecules are synthesized in an alternating 5′-3′, 3′-5′manner, such that they base pair with first one strand of a duplex andthen the other, eliminating the necessity for a sizeable stretch ofeither purines or pyrimidines to be present on one strand of a duplex.

[0127] The invention also provides detectably labeled oligonucleotideprimer and probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

[0128] A 53014 nucleic acid molecule can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup et al. (1996)Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms“peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., aDNA mimic, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength. Thesynthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described in Hyrup et al. (1996) supra;Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. 93: 14670-675.

[0129] PNAs of 53014 nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of 53014 nucleic acid molecules can also beused in the analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., S1 nucleases (Hyrup etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup et al. (1996) supra; Perry-O'Keefe supra).

[0130] In other embodiments, the oligonucleotide can include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier(see, e.g., PCT Publication No. W089/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents. (see, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide can be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

[0131] The invention also includes molecular beacon oligonucleotideprimer and probe molecules having at least one region which iscomplementary to a 53014 nucleic acid of the invention, twocomplementary regions one having a fluorophore and one a quencher suchthat the molecular beacon is useful for quantitating the presence of the53014 nucleic acid of the invention in a sample. Molecular beaconnucleic acids are described, for example, in Lizardi et al., U.S. Pat.No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livak etal., U.S. Pat. No. 5,876,930.

[0132] Isolated 53014 Polypeptides

[0133] In another aspect, the invention features, an isolated 53014protein, or fragment, e.g., a biologically active portion, for use asimmunogens or antigens to raise or test (or more generally to bind)anti-53014 antibodies. 53014 protein can be isolated from cells ortissue sources using standard protein purification techniques. 53014protein or fragments thereof can be produced by recombinant DNAtechniques or synthesized chemically.

[0134] Polypeptides of the invention include those which arise as aresult of the existence of multiple genes, alternative transcriptionevents, alternative RNA splicing events, and alternative translationaland post-translational events. The polypeptide can be expressed insystems, e.g., cultured cells, which result in substantially the samepost-translational modifications present when the polypeptide isexpressed in a native cell, or in systems which result in the alterationor omission of post-translational modifications, e.g., glycosylation orcleavage, present in a native cell.

[0135] In a preferred embodiment, a 53014 polypeptide has one or more ofthe following characteristics:

[0136] it has the ability to bind a metal atom or ion, e.g. zinc,calcium, manganese or magnesium;

[0137] it has the ability to bind a macromolecule, e.g. an extracellularmatrix molecule or protein (e.g. collagen, fibronectin, gelatin,vitronectin, thrombospondin, aggrecan, fibrinogen, fibrin, heparansulfate, dermatan sulfate, chondroitin sulfate, bone sialoprotein,elastin or laminin);

[0138] it has the ability to modulate the propeptide and convert fromzymogen to active enzyme;

[0139] it has the ability to cleave a protein by hydrolysis of an amidebond in a protein;

[0140] it has the ability to modulate cell motility/migration;

[0141] it has a molecular weight, e.g., a deduced molecular weight,preferably ignoring any contribution of post translationalmodifications, amino acid composition or other physical characteristicof a 53014 polypeptide, e.g., a polypeptide of SEQ ID NO: 2 or SEQ IDNO: 5;

[0142] it has an overall sequence similarity of at least 60%, preferablyat least 70%, more preferably at least 80, 90, or 95%, with apolypeptide of SEQ ID NO: 2 or SEQ ID NO: 5;

[0143] it can be found in brain cortex, spinal cord, hypothalamus, colonadenocarcinoma, lung tumor, ovary tumor, primary osteoblasts, erythroidcells and the K562 erythroid cell line;

[0144] it has a reprolysin family propeptide domain which is preferablyabout 70%, 80%, 90% or 95% identical to amino acid residues about 120 to240 of SEQ ID NO: 2; or amino acids 53 to 173 of SEQ ID NO: 5;

[0145] it has a reprolysin domain which is preferably about 70%, 80%,90% or 95% identical to amino acid residues about 261 to 460 of SEQ IDNO: 2 or 194 to 393 of SEQ ID NO: 5;

[0146] it has a thrombospondin domain which is preferably about 70%,80%, 90% or 95% identical to amino acid residues about 556 to 606, 849to 906, 911 to 968, or 970 to 1021 of SEQ ID NO: 2 or 489 to 539, 782 to839, 844 to 901, or 903 to 954 of SEQ ID NO: 5;

[0147] In a preferred embodiment the 53014 protein, or fragment thereof,differs from the corresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 5.In one embodiment it differs by at least one but by less than 15, 10 or5 amino acid residues. In another it differs from the correspondingsequence in SEQ ID NO: 2 or SEQ ID NO: 5 by at least one residue butless than 20%, 15%, 10% or 5% of the residues in it differ from thecorresponding sequence in SEQ ID NO: 2 or SEQ ID NO: 5. (If thiscomparison requires alignment the sequences should be aligned formaximum homology. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.) The differences are,preferably, differences or changes at a non-essential residue or aconservative substitution. In a preferred embodiment the differences arenot in the reprolysin family propeptide domain about amino acid residues120 to 240 of SEQ ID NO: 2 or amino acids 53 to 173 of SEQ ID NO: 5; orthe reprolysin domain about amino acid residues 261 to 460 of SEQ ID NO:2 or 194 to 393 of SEQ ID NO: 5; or a thrombospondin domain about aminoacid residues 556 to 606, 849 to 906, 911 to 968, or 970 to 1021 of SEQID NO: 2 or 489 to 539, 782 to 839, 844 to 901, or 903 to 954 of SEQ IDNO: 5. In another embodiment one or more differences are in thereprolysin family propeptide domain about amino acid residues 120 to 240of SEQ ID NO: 2 or amino acids 53 to 173 of SEQ ID NO: 5; or thereprolysin domain about amino acid residues 261 to 460 of SEQ II NO: 2or 194 to 393 of SEQ ID NO: 5; or a thrombospondin domain about aminoacid residues 556 to 606, 849 to 906, 911 to 968, or 970 to 1021 of SEQID NO: 2 or 489 to 539, 782 to 839, 844 to 901, or 903 to 954 of SEQ IDNO: 5.

[0148] Other embodiments include a protein that contains one or morechanges in amino acid sequence, e.g., a change in an amino acid residuewhich is not essential for activity. Such 53014 proteins differ in aminoacid sequence from SEQ ID NO: 2 or SEQ ID NO: 5, yet retain biologicalactivity.

[0149] In one embodiment, the protein includes an amino acid sequence atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or morehomologous to SEQ ID NO: 2 or SEQ ID NO: 5. In another embodiment, theprotein includes fragments or regions homologous to fragments, at leastabout 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to afragment of SEQ ID NO: 2 or SEQ ID NO: 5. A fragment of a 53014 proteincan be a domain, e.g. a reprolysin domain about amino acid residues 261to 460 of SEQ ID NO: 2 or 194 to 393 of SEQ ID NO: 5, or a fragmentthereof, e.g. about amino acid residues 261 to 300, 301 to 375, or 376to 460 of SEQ ID NO: 2, a reprolysin family propeptide domain aboutamino acid residues 120 to 240 of SEQ ID NO: 2; or amino acids 53 to 173of SEQ ID NO: 5, or a thrombospondin domain about amino acid residues556 to 606, 849 to 906, 911 to 968, or 970 to 1021 of SEQ ID NO: 2 or489 to 539, 782 to 839, 844 to 901, or 903 to 954 of SEQ ID NO: 5.

[0150] A 53014 protein or fragment is provided which varies from thesequence of SEQ ID NO: 2 in regions defined by amino acids about 1 to119, 241 to 259, 461 to 555, 607 to 848, 907 to 910, or 1022 to 1223 orfrom the sequence of SEQ ID NO: 5 in regions defined by amino acidsabout 1 to 52, 174 to 193, 394 to 488, 540 to 781, 840 to 843, or 955 to1156 by at least one but by less than 15, 10 or 5 amino acid residues inthe protein or fragment but which does not differ from SEQ ID NO: 2 inregions defined by amino acids about 261 to 460, 120 to 240, 556 to 606,849 to 906, 911 to 968, or 970 to 1021 or from 53 to 173, 194 to 393,489 to 539, 782 to 839, 844 to 901, or 903 to 954 of SEQ ID NO: 5.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences.) In some embodiments the difference is at anon-essential residue or is a conservative substitution, while in othersthe difference is at an essential residue or is a non-conservativesubstitution.

[0151] In one embodiment, a biologically active portion of a 53014protein includes a reprolysin domain, a reprolysin family propeptidedomain, or a thrombospondin domain. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native 53014 protein.

[0152] In a preferred embodiment, the 53014 protein has an amino acidsequence shown in SEQ ID NO: 2 or SEQ ID NO: 5. In other embodiments,the 53014 protein is sufficiently or substantially identical to SEQ IDNO: 2 or SEQ ID NO: 5. In yet another embodiment, the 53014 protein issufficiently or substantially identical to SEQ ID NO: 2 or SEQ ID NO: 5and retains the functional activity of the protein of SEQ ID NO: 2 orSEQ ID NO: 5, as described in detail in the subsections above.

[0153] 53014 Chimeric or Fusion Proteins

[0154] In another aspect, the invention provides 53014 chimeric orfusion proteins. As used herein, a 53014 “chimeric protein” or “fusionprotein” includes a 53014 polypeptide linked to a non-53014 polypeptide.A “non-53014 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the 53014 protein, e.g., a protein which is different fromthe 53014 protein and which is derived from the same or a differentorganism. The 53014 polypeptide of the fusion protein can correspond toall or a portion e.g., a fragment described herein of a 53014 amino acidsequence. In a preferred embodiment, a 53014 fusion protein includes atleast one (or two) biologically active portion of a 53014 protein. Thenon-53014 polypeptide can be fused to the N-terminus or C-terminus ofthe 53014 polypeptide.

[0155] The fusion protein can include a moiety which has a high affinityfor a ligand. For example, the fusion protein can be a GST-53014 fusionprotein in which the 53014 sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant 53014. Alternatively, the fusion protein can be a 53014protein containing a heterologous signal sequence at its N-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of 53014 can be increased through use of a heterologous signalsequence.

[0156] Fusion proteins can include all or a part of a serum protein,e.g., a portion of an immunoglobulin (e.g., IgG, IgA, or IgE), e.g., anFc region and/or the hinge C2 and C2 sequences of an immunoglobulin orhuman serum albumin.

[0157] The 53014 fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo.The 53014 fusion proteins can be used to affect the bioavailability of a53014 substrate. 53014 fusion proteins can be useful therapeutically forthe treatment of disorders caused by, for example, (i) aberrantmodification or mutation of a gene encoding a 53014 protein; (ii)mis-regulation of the 53014 gene; and (iii) aberrant post-translationalmodification of a 53014 protein.

[0158] Moreover, the 53014-fusion proteins of the invention can be usedas immunogens to produce anti-53014 antibodies in a subject, to purify53014 ligands and in screening assays to identify molecules whichinhibit the interaction of 53014 with a 53014 substrate.

[0159] Expression vectors are commercially available that already encodea fusion moiety (e.g., a GST polypeptide). A 53014-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the 53014 protein.

[0160] Variants of 53014 Proteins

[0161] In another aspect, the invention also features a variant of a53014 polypeptide, e.g., which functions as an agonist (mimetics) or asan antagonist. Variants of the 53014 proteins can be generated bymutagenesis, e.g., discrete point mutation, the insertion or deletion ofsequences or the truncation of a 53014 protein. An agonist of the 53014proteins can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of a 53014protein. An antagonist of a 53014 protein can inhibit one or more of theactivities of the naturally occurring form of the 53014 protein by, forexample, competitively modulating a 53014-mediated activity of a 53014protein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Preferably, treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein has fewer side effects in asubject relative to treatment with the naturally occurring form of the53014 protein.

[0162] Variants of a 53014 protein can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a 53014protein for agonist or antagonist activity.

[0163] Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a 53014 protein coding sequence can be used to generate avariegated population of fragments for screening and subsequentselection of variants of a 53014 protein.

[0164] Variants in which a cysteine residues is added or deleted or inwhich a residue which is glycosylated is added or deleted areparticularly preferred.

[0165] Methods for screening gene products of combinatorial librariesmade by point mutations or truncation, and for screening cDNA librariesfor gene products having a selected property are known in the art.Recursive ensemble mutagenesis (REM), a new technique which enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify 53014 variants (Arkinand Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave etal. (1993) Protein Engineering 6:327-331).

[0166] Cell based assays can be exploited to analyze a variegated 53014library. For example, a library of expression vectors can be transfectedinto a cell line, e.g., a cell line, which ordinarily responds to 53014in a substrate-dependent manner. The transfected cells are thencontacted with 53014 and the effect of the expression of the mutant onsignaling by the 53014 substrate can be detected, e.g., by measuring thebinding of a metal atom or ion, e.g. zinc, calcium, manganese ormagnesium or the binding or catalyzing the cleavage of a macromolecule,e.g. an extracellular matrix molecule or protein (e.g. collagen,fibronectin, gelatin, vitronectin, thrombospondin, aggrecan, fibrinogen,fibrin, heparan sulfate, dermatan sulfate, chondroitin sulfate, bonesialoprotein, elastin or laminin). Plasmid DNA can then be recoveredfrom the cells which score for inhibition, or alternatively,potentiation of signaling by the 53014 substrate, and the individualclones further characterized.

[0167] Activity assays for metalloprotease family members, such as 53014polypeptides, involve any of the known metalloprotease, reprolysin, orthrombospondin-like activity or functions, as well asactivities/functions that may not typically be found in othermetalloproteases. These assays include, but are not limited to, assayswhich measure the binding of a 53014 molecule to other molecules oratoms, e.g. the extracellular matrix, collagen or gelatin, integrin,zinc or other metals, or α2-macroglobulin; assays which measure theability of a 53014 molecule to cleave specific peptide substrates toproduce fragments or to break down cartilage; assays which measure theability of a 53014 molecule to affect cell-cell adhesion or cell-matrixinteraction, e.g. the binding of integrin or heparin or other sulfatedglycosaminoglycan, (e.g. heparan sulfate); assays which measure theability of a 53014 molecule to modulate vascularization or vascularendothelial growth, inducing apoptosis of endothelial cells; assayswhich measure the ability of a 53014 molecule to suppress tumor growth,e.g. to modulate angiogenesis; assays which measure the ability of a53014 molecule to affect cellular chemotaxis; and assays which measurethe ability of a 53014 molecule to affect any of the other biological orfunctional properties of these proteins, including, but not limited to,those disclosed herein, and in the references cited herein. Further,assays can relate to changes in the 53014 protein, per se, and on theeffects of these changes, for example, cleavage of the substrate,activation of the protein following cleavage, etc.

[0168] Such assays are described in Tang et al. (1999) FEBS Letters445:223-225 (for example, induction by interleukin I in vitro and byintravenous administration of lipopolysaccharide in vivo, as well aseffects on cell adhesion, motility, and growth); Abbaszade et al., supra(for example, products resulting from cleavage at the Glu-Ala site incartilage explants and chondrocyte cultures treated with interleukin Iand retinoic acid, determination of aggrecan cleaving activity with andwithout hydroxamate inhibitors); Kuno et al. (1998), supra (binding tothe extracellular matrix, binding to sulfated glycosaminoglycans,binding to heparan sulfate); Kuno et al. (1999) (protease trapping ofa2-macroglobulin, furin processing); Tortorella et al. (1999), supra(detection of aggrecan fragments, especially by neoepitope antibodies,inhibition of cleavage by ADAMTS inhibitors, inhibition of pro-MMPactivation); Vasquez et al., supra (suppression of fibroblast growthfactor-2-induced vascularization in the cornea pocket assay andinhibition of vascular endothelial growth factor-induced angiogenesis inthe chorioallantoic membrane assay, inhibition of endothelial cellproliferation, competitive inhibition with endostatin, proliferation ofhuman dermal endothelial cells, use of the antiangiogenic region of theTSP-1 motif as bait); Kuno et al. (1997), supra (heparin binding,induction of expression in vitro by interleukin I, induction ofexpression in vivo by LPS); Wolfsberg et al., supra (degradation ofbasement membrane, binding of integrin, and fusogenic activity); Guilpinet al. (1988) J. Biol. Chem. 273:157-166 (α2-macroglobulin trapping,cleavage of prodomain at the furin site to generate activemetalloprotease); Rosendahl et al. (1997) J. Biol. Chem.272:24588-24593, TNF α processing). Recombinant assay systems include,but are not limited to, those described in Abbaszade et al., supra; Kunoet al. (1998), supra; Kuno et al. (1999), supra; Tortorella et al.,supra; Vasquez et al., supra, and Kuno et al. (1997), supra.

[0169] In another aspect, the invention features a method of making a53014 polypeptide, e.g., a peptide having a non-wild type activity,e.g., an antagonist, agonist, or super agonist of a naturally occurring53014 polypeptide, e.g., a naturally occurring 53014 polypeptide. Themethod includes altering the sequence of a 53014 polypeptide, e.g.,altering the sequence, e.g., by substitution or deletion of one or moreresidues of a non-conserved region, a domain or residue disclosedherein, and testing the altered polypeptide for the desired activity.

[0170] In another aspect, the invention features a method of making afragment or analog of a 53014 polypeptide a biological activity of anaturally occurring 53014 polypeptide. The method includes altering thesequence, e.g., by substitution or deletion of one or more residues, ofa 53014 polypeptide, e.g., altering the sequence of a non-conservedregion, or a domain or residue described herein, and testing the alteredpolypeptide for the desired activity.

[0171] Anti-53014 Antibodies

[0172] In another aspect, the invention provides an anti-53014 antibody.The term “antibody” as used herein refers to an immunoglobulin moleculeor immunologically active portion thereof, i.e., an antigen-bindingportion. Examples of immunologically active portions of immunoglobulinmolecules include scFV and dcFV fragments, Fab and F(ab′)₂ fragmentswhich can be generated by treating the antibody with an enzyme such aspapain or pepsin, respectively.

[0173] The antibody can be a polyclonal, monoclonal, recombinant, e.g.,a chimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

[0174] A full-length 53014 protein or, antigenic peptide fragment of53014 can be used as an immunogen or can be used to identify anti-53014antibodies made with other immunogens, e.g., cells, membranepreparations, and the like. The antigenic peptide of 53014 shouldinclude at least 8 amino acid residues of the amino acid sequence shownin SEQ ID NO: 2 or SEQ ID NO: 5 and encompasses an epitope of 53014.Preferably, the antigenic peptide includes at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

[0175] Fragments of 53014 which include residues about 242 to 253, fromabout 573 to 581, and from about 889 to 904 of SEQ ID NO: 2 can be usedto make, e.g., used as immunogens or used to characterize thespecificity of an antibody, antibodies against hydrophilic regions ofthe 53014 protein (see FIG. 1). Similarly, fragments of 53014 whichinclude residues about 56 to 70, from about 155 to 168, and from about300 to 315 of SEQ ID NO: 2 can be used to make an antibody against ahydrophobic region of the 53014 protein; fragments of 53014 whichinclude residues about, 1 to 1223 or a subset thereof, e.g. aboutresidues 1 to 22, 23 to 50, 51 to 120, 1022 to 1050, 1051 to 1100, 1101to 1150, 1151 to 1190 or 1191 to 1223 of SEQ ID NO: 2 can be used tomake an antibody against an extracellular region of the 53014 protein; afragment of 53014 which include residues about 261 to 460 of SEQ ID NO:2, or a subset thereof, e.g. 261 to 325, 326 to 400 or 401 or 460 of SEQID NO: 2 can be used to make an antibody against the reprolysin regionof the 53014 protein; a fragment of 53014 which include residues about120 to 240 of SEQ ID NO: 2, can be used to make an antibody against thereprolysin family propeptide region of the 53014 protein; a fragment of53014 which include residues about 556 to 606, 849 to 906, 911 to 968,and 970 to 1021 of SEQ ID NO: 2, can be used to make an antibody againsta thrombospondin region of the 53014 protein.

[0176] Fragments of 53014 which include residues about 1 to 35, fromabout 170 to 190, and from about 800 to 840 of SEQ ID NO: 5 can be usedto make, e.g., used as immunogens or used to characterize thespecificity of an antibody, antibodies against hydrophilic regions ofthe 53014 protein. Similarly, fragments of 53014 which include residuesabout 89 to 111, or about 233 to 249 of SEQ ID NO: 5 can be used to makean antibody against a hydrophobic region of the 53014 protein; afragment of 53014 which include residues about 53 to 173, 194 to 363,and 489 to 539, 782 to 839, 844 to 901, 903 to 954 of SEQ ID NO: 5 canbe used to make an antibody against the propeptide domain, themetalloprotease domain and one or more of the thrombospondin domains,respectively. In another embodiment fragments can have one or moredifferences in the regions described above.

[0177] Antibodies reactive with, or specific or selective for, any ofthese regions, or other regions or domains described herein areprovided.

[0178] Preferred epitopes encompassed by the antigenic peptide areregions of 53014 located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity. Forexample, an Emini surface probability analysis of the human 53014protein sequence can be used to indicate the regions that have aparticularly high probability of being localized to the surface of the53014 protein and are thus likely to constitute surface residues usefulfor targeting antibody production.

[0179] In a preferred embodiment the antibody binds an epitope on anydomain or region on 53014 proteins described herein.

[0180] Additionally, chimeric, humanized, and completely humanantibodies are also within the scope of the invention. Chimeric,humanized, but most preferably, completely human antibodies aredesirable for applications which include repeated administration, e.g.,therapeutic treatment of human patients, and some diagnosticapplications.

[0181] Chimeric and humanized monoclonal antibodies, comprising bothhuman and non-human portions, can be made using standard recombinant DNAtechniques. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in Robinson et al. International Application No.PCT/US86/02269; Akira, et al. European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al. EuropeanPatent Application 173,494; Neuberger et al. PCT InternationalPublication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567;Cabilly et al. European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559).

[0182] A humanized or complementarity determining region (CDR)-graftedantibody will have at least one or two, but generally all threerecipient CDR's (of heavy and or light immuoglobulin chains) replacedwith a donor CDR. The antibody may be replaced with at least a portionof a non-human CDR or only some of the CDR's may be replaced withnon-human CDR's. It is only necessary to replace the number of CDR'srequired for binding of the humanized antibody to a 53014 or a fragmentthereof. Preferably, the donor will be a rodent antibody, e.g., a rat ormouse antibody, and the recipient will be a human framework or a humanconsensus framework. Typically, the immunoglobulin providing the CDR'sis called the “donor” and the immunoglobulin providing the framework iscalled the “acceptor.” In one embodiment, the donor immunoglobulin is anon-human (e.g., rodent). The acceptor framework is anaturally-occurring (e.g., a human) framework or a consensus framework,or a sequence about 85% or higher, preferably 90%, 95%, 99% or higheridentical thereto.

[0183] As used herein, the term “consensus sequence” refers to thesequence formed from the most frequently occurring amino acids (ornucleotides) in a family of related sequences (See e.g., Winnaker,(1987) From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany). Ina family of proteins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence. A “consensus framework” refers tothe framework region in the consensus immunoglobulin sequence.

[0184] An antibody can be humanized by methods known in the art.Humanized antibodies can be generated by replacing sequences of the Fvvariable region which are not directly involved in antigen binding withequivalent sequences from human Fv variable regions. General methods forgenerating humanized antibodies are provided by Morrison (1985) Science229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and by Queen etal. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents ofall of which are hereby incorporated by reference. Those methods includeisolating, manipulating, and expressing the nucleic acid sequences thatencode all or part of immunoglobulin Fv variable regions from at leastone of a heavy or light chain. Sources of such nucleic acid are wellknown to those skilled in the art and, for example, may be obtained froma hybridoma producing an antibody against a 53014 polypeptide orfragment thereof. The recombinant DNA encoding the humanized antibody,or fragment thereof, can then be cloned into an appropriate expressionvector.

[0185] Humanized or CDR-grafted antibodies can be produced byCDR-grafting or CDR substitution, wherein one, two, or all CDR's of animmunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539;Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science239:1534; Beidler et al. (1988) J. Immunol. 141:4053-4060; Winter U.S.Pat. No. 5,225,539, the contents of all of which are hereby expresslyincorporated by reference. Winter describes a CDR-grafting method whichmay be used to prepare the humanized antibodies of the present invention(UK Patent Application GB 88638A, filed on Mar. 26, 1987; Winter U.S.Pat. No. 5,225,539), the contents of which is expressly incorporated byreference.

[0186] Also within the scope of the invention are humanized antibodiesin which specific amino acids have been substituted, deleted or added.Preferred humanized antibodies have amino acid substitutions in theframework region, such as to improve binding to the antigen. Forexample, a humanized antibody will have framework residues identical tothe donor framework residue or to another amino acid other than therecipient framework residue. To generate such antibodies, a selected,small number of acceptor framework residues of the humanizedimmunoglobulin chain can be replaced by the corresponding donor aminoacids. Preferred locations of the substitutions include amino acidresidues adjacent to the CDR, or which are capable of interacting with aCDR (see e.g., U.S. Pat. No. 5,585,089). Criteria for selecting aminoacids from the donor are described in U.S. Pat. No. 5,585,089, e.g.,columns 12-16 of U.S. Pat. No. 5,585,089, the e.g., columns 12-16 ofU.S. Pat. No. 5,585,089, the contents of which are hereby incorporatedby reference. Other techniques for humanizing antibodies are describedin Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

[0187] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Such antibodies can be producedusing transgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), canbe engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

[0188] Completely human antibodies that recognize a selected epitope canbe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

[0189] The anti-53014 antibody can be a single chain antibody. Asingle-chain antibody (scFV) can be engineered as described in, forexample, Colcher et al. (1999) Ann. N Y Acad. Sci. 880:263-80; andReiter (1996) Clin. Cancer Res. 2:245-52. The single chain antibody canbe dimerized or multimerized to generate multivalent antibodies havingspecificities for different epitopes of the same target 53014 protein.

[0190] In a preferred embodiment, the antibody has reduced or no abilityto bind an Fc receptor. For example, it is an isotype or subtype,fragment or other mutant, which does not support binding to an Fcreceptor, e.g., it has a mutagenized or deleted Fc receptor bindingregion.

[0191] An antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive ion. A cytotoxin or cytotoxic agent includes any agent thatis detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g.,maytansinol (see U.S. Pat. No. 5,208,020), CC-1065 (see U.S. Pat. Nos.5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof.Therapeutic agents include, but are not limited to, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactiveions include, but are not limited to iodine, yttrium and praseodymium.

[0192] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-interferon, β-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

[0193] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980.

[0194] An anti-53014 antibody (e.g., monoclonal antibody) can be used toisolate 53014 by standard techniques, such as affinity chromatography orimmunoprecipitation. Moreover, an anti-53014 antibody can be used todetect 53014 protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theprotein. Anti-53014 antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance (i.e., antibody labelling). Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0195] In preferred embodiments, an antibody can be made by immunizingwith a purified 53014 antigen, or a fragment thereof, e.g., a fragmentdescribed herein, a membrane associated antigen, tissues, e.g., crudetissue preparations, whole cells, preferably living cells, lysed cells,or cell fractions, e.g., membrane fractions, or cell culturesupernatants, e.g., conditioned medium.

[0196] Antibodies which bind only a native 53014 protein, only denaturedor otherwise non-native 53014 protein, or which bind both, are withinthe invention. Antibodies with linear or conformational epitopes arewithin the invention. Conformational epitopes sometimes can beidentified by identifying antibodies which bind to native but notdenatured 53014 protein.

[0197] Recombinant Expression Vectors, Host Cells and GeneticallyEngineered Cells

[0198] In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

[0199] A vector can include a 53014 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Preferably therecombinant expression vector includes one or more regulatory sequencesoperatively linked to the nucleic acid sequence to be expressed. Theterm “regulatory sequence” includes promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those which direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired, and the like. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or polypeptides, including fusion proteins or polypeptides,encoded by nucleic acids as described herein (e.g., 53014 proteins,mutant forms of 53014 proteins, fusion proteins, and the like).

[0200] The recombinant expression vectors of the invention can bedesigned for expression of 53014 proteins in prokaryotic or eukaryoticcells. For example, polypeptides of the invention can be expressed in E.coli, insect cells (e.g., using baculovirus expression vectors), yeastcells or mammalian cells. Suitable host cells are discussed further inGoeddel, (1990) Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0201] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein.

[0202] Purified fusion proteins can be used in 53014 activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific or selective for 53014 proteins. In apreferred embodiment, a fusion protein expressed in a retroviralexpression vector of the present invention can be used to infect bonemarrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six weeks).

[0203] To maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman (1990) GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. 119-128). Another strategy is to alter the nucleic acidsequence of the nucleic acid to be inserted into an expression vector sothat the individual codons for each amino acid are those preferentiallyutilized in E. coli (Wada et al., (1992) Nucleic Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

[0204] The 53014 expression vector can be a yeast expression vector, avector for expression in insect cells, e.g., a baculovirus expressionvector or a vector suitable for expression in mammalian cells.

[0205] When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40.

[0206] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid). Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0207] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub et al., (1986) Reviews—Trends inGenetics 1:1.

[0208] Another aspect the invention provides a host cell which includesa nucleic acid molecule described herein, e.g., a 53014 nucleic acidmolecule within a recombinant expression vector or a 53014 nucleic acidmolecule containing sequences which allow it to homologously recombineinto a specific site of the host cell's genome. The terms “host cell”and “recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but also to the progeny orpotential progeny of such a cell. Because certain modifications canoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0209] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a 53014 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary (CHO) cells or CV-1 origin, SV-40 (COS) cells). Other suitablehost cells are known to those skilled in the art.

[0210] Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

[0211] A host cell of the invention can be used to produce (i.e.,express) a 53014 protein. Accordingly, the invention further providesmethods for producing a 53014 protein using the host cells of theinvention. In one embodiment, the method includes culturing the hostcell of the invention (into which a recombinant expression vectorencoding a 53014 protein has been introduced) in a suitable medium suchthat a 53014 protein is produced. In another embodiment, the methodfurther includes isolating a 53014 protein from the medium or the hostcell.

[0212] In another aspect, the invention features, a cell or purifiedpreparation of cells which include a 53014 transgene, or which otherwisemisexpress 53014. The cell preparation can consist of human or non-humancells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, orpig cells. In preferred embodiments, the cell or cells include a 53014transgene, e.g., a heterologous form of a 53014, e.g., a gene derivedfrom humans (in the case of a non-human cell). The 53014 transgene canbe misexpressed, e.g., overexpressed or underexpressed. In otherpreferred embodiments, the cell or cells include a gene whichmisexpresses an endogenous 53014, e.g., a gene the expression of whichis disrupted, e.g., a knockout. Such cells can serve as a model forstudying disorders which are related to mutated or misexpressed 53014alleles or for use in drug screening.

[0213] In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject 53014 polypeptide.

[0214] Also provided are cells, preferably human cells, e.g., humanhematopoietic or fibroblast cells, in which an endogenous 53014 is underthe control of a regulatory sequence that does not normally control theexpression of the endogenous 53014 gene. The expression characteristicsof an endogenous gene within a cell, e.g., a cell line or microorganism,can be modified by inserting a heterologous DNA regulatory element intothe genome of the cell such that the inserted regulatory element isoperably linked to the endogenous 53014 gene. For example, an endogenous53014 gene which is “transcriptionally silent,” e.g., not normallyexpressed, or expressed only at very low levels, can be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell. Techniquessuch as targeted homologous recombinations, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published in May 16, 1991.

[0215] Transgenic Animals

[0216] The invention provides non-human transgenic animals. Such animalsare useful for studying the function and/or activity of a 53014 proteinand for identifying and/or evaluating modulators of 53014 activity. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, amphibians, and the like. A transgene is exogenous DNA or arearrangement, e.g., a deletion of endogenous chromosomal DNA, whichpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A transgene can direct the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal, other transgenes, e.g., a knockout, reduce expression. Thus, atransgenic animal can be one in which an endogenous 53014 gene has beenaltered by, e.g., by homologous recombination between the endogenousgene and an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

[0217] Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a transgene of the invention in order to direct expression ofa 53014 protein to particular cells. A transgenic founder animal can beidentified based upon the presence of a 53014 transgene in its genomeand/or expression of 53014 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a 53014 protein can further be bred to othertransgenic animals carrying other transgenes.

[0218] 53014 proteins or polypeptides can be expressed in transgenicanimals or plants, e.g., a nucleic acid encoding the protein orpolypeptide can be introduced into the genome of an animal. In preferredembodiments the nucleic acid is placed under the control of a tissuespecific promoter, e.g., a milk or egg specific promoter, and recoveredfrom the milk or eggs produced by the animal. Suitable animals are mice,pigs, cows, goats, and sheep.

[0219] The invention also includes a population of cells from atransgenic animal, as discussed, e.g., below.

[0220] Uses

[0221] The nucleic acid molecules, proteins, protein homologs, andantibodies described herein can be used in one or more of the followingmethods: a) screening assays; b) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenetics); and c) methods of treatment (e.g., therapeutic andprophylactic).

[0222] The isolated nucleic acid molecules of the invention can be used,for example, to express a 53014 protein (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect a 53014 mRNA (e.g., in a biological sample) or a geneticalteration in a 53014 gene, and to modulate 53014 activity, as describedfurther below. The 53014 proteins can be used to treat disorderscharacterized by insufficient or excessive production of a 53014substrate or production of 53014 inhibitors. In addition, the 53014proteins can be used to screen for naturally occurring 53014 substrates,to screen for drugs or compounds which modulate 53014 activity, as wellas to treat disorders characterized by insufficient or excessiveproduction of 53014 protein or production of 53014 protein forms whichhave decreased, aberrant or unwanted activity compared to 53014 wildtype protein (e.g., aberrant or deficient metalloprotease, e.g.reprolysin (M12B) (e.g. ADAMTS) function or expression). Moreover, theanti-53014 antibodies of the invention can be used to detect and isolate53014 proteins, regulate the bioavailability of 53014 proteins, andmodulate 53014 activity.

[0223] A method of evaluating a compound for the ability to interactwith, e.g., bind, a subject 53014 polypeptide is provided. The methodincludes: contacting the compound with the subject 53014 polypeptide;and evaluating ability of the compound to interact with, e.g., to bindor form a complex with the subject 53014 polypeptide. This method can beperformed in vitro, e.g., in a cell free system, or in vivo, e.g., in atwo-hybrid interaction trap assay. This method can be used to identifynaturally occurring molecules which interact with subject 53014polypeptide. It can also be used to find natural or synthetic inhibitorsof subject 53014 polypeptide. Screening methods are discussed in moredetail below.

[0224] Screening Assays

[0225] The invention provides methods (also referred to herein as“screening assays”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., proteins, peptides, peptidomimetics,peptoids, small molecules or other drugs) which bind to 53014 proteins,have a stimulatory or inhibitory effect on, for example, 53014expression or 53014 activity, or have a stimulatory or inhibitory effecton, for example, the expression or activity of a 53014 substrate.Compounds thus identified can be used to modulate the activity of targetgene products (e.g., 53014 genes) in a therapeutic protocol, toelaborate the biological function of the target gene product, or toidentify compounds that disrupt normal target gene interactions.

[0226] In one embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a 53014 protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of a 53014 proteinor polypeptide or a biologically active portion thereof.

[0227] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including: biological libraries; peptoid libraries (librariesof molecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994)J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

[0228] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. U.S.A. 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci.USA 91:11422-426; Zuckermann et al. (1994). J. Med. Chem. 37:2678-85;Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233-51.

[0229] Libraries of compounds can be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[0230] In one embodiment, an assay is a cell-based assay in which a cellwhich expresses a 53014 protein or biologically active portion thereofis contacted with a test compound, and the ability of the test compoundto modulate 53014 activity is determined. Determining the ability of thetest compound to modulate 53014 activity can be accomplished bymonitoring, for example, metalloprotease, e.g. reprolysin (M12B) (e.g.ADAMTS) function. The cell, for example, can be of mammalian origin,e.g., human.

[0231] The ability of the test compound to modulate 53014 binding to acompound, e.g., a 53014 substrate, or to bind to 53014 can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to 53014 can bedetermined by detecting the labeled compound, e.g., substrate, in acomplex. Alternatively, 53014 could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulate53014 binding to a 53014 substrate in a complex. For example, compounds(e.g., 53014 substrates) can be labeled with ¹²⁵I, ¹⁴C, ³⁵S or ³H.,either directly or indirectly, and the radioisotope detected by directcounting of radioemmission or by scintillation counting. Alternatively,compounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

[0232] The ability of a compound (e.g., a 53014 substrate) to interactwith 53014 with or without the labeling of any of the interactants canbe evaluated. For example, a microphysiometer can be used to detect theinteraction of a compound with 53014 without the labeling of either thecompound or the 53014. McConnell et al. (1992) Science 257:1906-1912. Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and 53014.

[0233] In yet another embodiment, a cell-free assay is provided in whicha 53014 protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to bind to the53014 protein or biologically active portion thereof is evaluated.Preferred biologically active portions of the 53014 proteins to be usedin assays of the present invention include fragments which participatein interactions with non-53014 molecules, e.g., fragments with highsurface probability scores.

[0234] Soluble and/or membrane-bound forms of isolated proteins (e.g.,53014 proteins or biologically active portions thereof) can be used inthe cell-free assays of the invention. When membrane-bound forms of theprotein are used, it may be desirable to utilize a solubilizing agent.Examples of such solubilizing agents include non-ionic detergents suchas n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0235] Cell-free assays involve preparing a reaction mixture of thetarget gene protein and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex that can be removed and/or detected.

[0236] The interaction between two molecules can also be detected, e.g.,using fluorescence energy transfer (FET) (see, for example, Lakowicz etal., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule can simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label can be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

[0237] In another embodiment, determining the ability of the 53014protein to bind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander andUrbaniczky (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr.Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA”detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the bindingsurface (indicative of a binding event) result in alterations of therefractive index of light near the surface (the optical phenomenon ofsurface plasmon resonance (SPR)), resulting in a detectable signal whichcan be used as an indication of real-time reactions between biologicalmolecules.

[0238] In one embodiment, the target gene product or the test substanceis anchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

[0239] It may be desirable to immobilize either 53014, an anti-53014antibody or its target molecule to facilitate separation of complexedfrom uncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to a53014 protein, or interaction of a 53014 protein with a target moleculein the presence and absence of a candidate compound, can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtiter plates, test tubes, and micro-centrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows one or both of the proteins to be bound to a matrix.For example, glutathione-S-transferase/53014 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or 53014 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of 53014binding or activity determined using standard techniques.

[0240] Other techniques for immobilizing either a 53014 protein or atarget molecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated 53014 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

[0241] In order to conduct the assay, the non-immobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific or selective for the immobilizedcomponent (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody).

[0242] In one embodiment, this assay is performed utilizing antibodiesreactive with 53014 protein or target molecules but which do notinterfere with binding of the 53014 protein to its target molecule. Suchantibodies can be derivatized to the wells of the plate, and unboundtarget or 53014 protein trapped in the wells by antibody conjugation.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the 53014 protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the 53014 protein or target molecule.

[0243] Alternatively, cell free assays can be conducted in a liquidphase. In such an assay, the reaction products are separated fromunreacted components, by any of a number of standard techniques,including but not limited to: differential centrifugation (see, forexample, Rivas and Minton (1993) Trends Biochem Sci 18:284-7);chromatography (gel filtration chromatography, ion-exchangechromatography); electrophoresis (see, e.g., Ausubel et al., eds. (1999)Current Protocols in Molecular Biology, J. Wiley, New York.); andimmunoprecipitation (see, for example, Ausubel et al., eds. (1999)Current Protocols in Molecular Biology, J. Wiley, New York). Such resinsand chromatographic techniques are known to one skilled in the art (see,e.g., Heegaard (1998) J Mol Recognit 11: 141-8; Hage and Tweed (1997) JChromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energytransfer can also be conveniently utilized, as described herein, todetect binding without further purification of the complex fromsolution.

[0244] In a preferred embodiment, the assay includes contacting the53014 protein or biologically active portion thereof with a knowncompound which binds 53014 to form an assay mixture, contacting theassay mixture with a test compound, and determining the ability of thetest compound to interact with a 53014 protein, wherein determining theability of the test compound to interact with a 53014 protein includesdetermining the ability of the test compound to preferentially bind to53014 or biologically active portion thereof, or to modulate theactivity of a target molecule, as compared to the known compound.

[0245] The target gene products of the invention can, in vivo, interactwith one or more cellular or extracellular macromolecules, such asproteins. For the purposes of this discussion, such cellular andextracellular macromolecules are referred to herein as “bindingpartners.” Compounds that disrupt such interactions can be useful inregulating the activity of the target gene product. Such compounds caninclude, but are not limited to molecules such as antibodies, peptides,and small molecules. The preferred target genes/products for use in thisembodiment are the 53014 genes herein identified. In an alternativeembodiment, the invention provides methods for determining the abilityof the test compound to modulate the activity of a 53014 protein throughmodulation of the activity of a downstream effector of a 53014 targetmolecule. For example, the activity of the effector molecule on anappropriate target can be determined, or the binding of the effector toan appropriate target can be determined, as previously described.

[0246] To identify compounds that interfere with the interaction betweenthe target gene product and its cellular or extracellular bindingpartner(s), a reaction mixture containing the target gene product andthe binding partner is prepared, under conditions and for a timesufficient, to allow the two products to form complex. In order to testan inhibitory agent, the reaction mixture is provided in the presenceand absence of the test compound. The test compound can be initiallyincluded in the reaction mixture, or can be added at a time subsequentto the addition of the target gene and its cellular or extracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe target gene product and the cellular or extracellular bindingpartner is then detected. The formation of a complex in the controlreaction, but not in the reaction mixture containing the test compound,indicates that the compound interferes with the interaction of thetarget gene product and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal target gene product can also be compared to complex formationwithin reaction mixtures containing the test compound and mutant targetgene product. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal target gene products.

[0247] These assays can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the target geneproduct or the binding partner onto a solid phase, and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants can bevaried to obtain different information about the compounds being tested.For example, test compounds that interfere with the interaction betweenthe target gene products and the binding partners, e.g., by competition,can be identified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

[0248] In a heterogeneous assay system, either the target gene productor the interactive cellular or extracellular binding partner, isanchored onto a solid surface (e.g., a microtiter plate), while thenon-anchored species is labeled, either directly or indirectly. Theanchored species can be immobilized by non-covalent or covalentattachments. Alternatively, an immobilized antibody specific orselective for the species to be anchored can be used to anchor thespecies to the solid surface.

[0249] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific or selective forthe initially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with, e.g., a labeled anti-Igantibody). Depending upon the order of addition of reaction components,test compounds that inhibit complex formation or that disrupt preformedcomplexes can be detected.

[0250] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific or selective for one of the bindingcomponents to anchor any complexes formed in solution, and a labeledantibody specific or selective for the other partner to detect anchoredcomplexes. Again, depending upon the order of addition of reactants tothe liquid phase, test compounds that inhibit complex or that disruptpreformed complexes can be identified.

[0251] In an alternate embodiment of the invention, a homogeneous assaycan be used. For example, a preformed complex of the target gene productand the interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

[0252] In yet another aspect, the 53014 proteins can be used as “baitproteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S.Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with 53014 (“53014-binding proteins” or “53014-bp”) and areinvolved in 53014 activity. Such 53014-bps can be activators orinhibitors of signals by the 53014 proteins or 53014 targets as, forexample, downstream elements of a 53014-mediated signaling pathway.

[0253] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a 53014 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 53014 protein can bethe fused to the activator domain.) If the “bait” and the “prey”proteins are able to interact, in vivo, forming a 53014-dependentcomplex, the DNA-binding and activation domains of the transcriptionfactor are brought into close proximity. This proximity allowstranscription of a reporter gene (e.g., lacZ) which is operably linkedto a transcriptional regulatory site responsive to the transcriptionfactor. Expression of the reporter gene can be detected and cellcolonies containing the functional transcription factor can be isolatedand used to obtain the cloned gene which encodes the protein whichinteracts with the 53014 protein.

[0254] In another embodiment, modulators of 53014 expression areidentified. For example, a cell or cell free mixture is contacted with acandidate compound and the expression of 53014 mRNA or protein evaluatedrelative to the level of expression of 53014 mRNA or protein in theabsence of the candidate compound. When expression of 53014 mRNA orprotein is greater in the presence of the candidate compound than in itsabsence, the candidate compound is identified as a stimulator of 53014mRNA or protein expression. Alternatively, when expression of 53014 mRNAor protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of 53014 mRNA or protein expression. Thelevel of 53014 mRNA or protein expression can be determined by methodsdescribed herein for detecting 53014 mRNA or protein.

[0255] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulating agentcan be identified using a cell-based or a cell free assay, and theability of the agent to modulate the activity of a 53014 protein can beconfirmed in vivo, e.g., in an animal such as an animal model foraberrant or deficient metalloprotease, e.g. reprolysin (M12B) (e.g.ADAMTS) function or expression.

[0256] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a 53014 modulating agent, an antisense 53014 nucleic acidmolecule, a 53014-specific antibody, or a 53014-binding partner) in anappropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

[0257] Detection Assays

[0258] Portions or fragments of the nucleic acid sequences identifiedherein can be used as polynucleotide reagents. For example, thesesequences can be used to: (i) map their respective genes on a chromosomee.g., to locate gene regions associated with genetic disease or toassociate 53014 with a disease; (ii) identify an individual from aminute biological sample (tissue typing); and (iii) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

[0259] Chromosome Mapping

[0260] The 53014 nucleotide sequences or portions thereof can be used tomap the location of the 53014 genes on a chromosome. This process iscalled chromosome mapping. Chromosome mapping is useful in correlatingthe 53014 sequences with genes associated with disease.

[0261] Briefly, 53014 genes can be mapped to chromosomes by preparingPCR primers (preferably 15-25 bp in length) from the 53014 nucleotidesequences. These primers can then be used for PCR screening of somaticcell hybrids containing individual human chromosomes. Only those hybridscontaining the human gene corresponding to the 53014 sequences willyield an amplified fragment.

[0262] A panel of somatic cell hybrids in which each cell line containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, can allow easy mapping ofindividual genes to specific human chromosomes. (D'Eustachio et al.(1983) Science 220:919-924).

[0263] Other mapping strategies e.g., in situ hybridization (describedin Fan et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries can be used to map53014 to a chromosomal location.

[0264] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York).

[0265] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0266] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inMcKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland et al. (1987)Nature, 325:783-787.

[0267] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the 53014 gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0268] Tissue Typing

[0269] 53014 sequences can be used to identify individuals frombiological samples using, e.g., restriction fragment length polymorphism(RFLP). In this technique, an individual's genomic DNA is digested withone or more restriction enzymes, the fragments separated, e.g., in aSouthern blot, and probed to yield bands for identification. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

[0270] Furthermore, the sequences of the present invention can also beused to determine the actual base-by-base DNA sequence of selectedportions of an individual's genome. Thus, the 53014 nucleotide sequencesdescribed herein can be used to prepare two PCR primers from the 5′ and3′ ends of the sequences. These primers can then be used to amplify anindividual's DNA and subsequently sequence it. Panels of correspondingDNA sequences from individuals, prepared in this manner, can provideunique individual identifications, as each individual will have a uniqueset of such DNA sequences due to allelic differences.

[0271] Allelic variation occurs to some degree in the coding regions ofthese sequences, and to a greater degree in the noncoding regions. Eachof the sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO: 1 or SEQ ID NO: 4 canprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO: 3 orSEQ ID NO: 6 are used, a more appropriate number of primers for positiveindividual identification would be 500-2,000.

[0272] If a panel of reagents from 53014 nucleotide sequences describedherein is used to generate a unique identification database for anindividual, those same reagents can later be used to identify tissuefrom that individual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0273] Use of Partial 53014 Sequences in Forensic Biology

[0274] DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

[0275] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO: 1 or SEQ ID NO: 4 (e.g.,fragments derived from the noncoding regions of SEQ ID NO: 1 or SEQ IDNO: 4 having a length of at least 20 bases, preferably at least 30bases) are particularly appropriate for this use.

[0276] The 53014 nucleotide sequences described herein can further beused to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue. This can be very useful incases where a forensic pathologist is presented with a tissue of unknownorigin. Panels of such 53014 probes can be used to identify tissue byspecies and/or by organ type.

[0277] In a similar fashion, these reagents, e.g., 53014 primers orprobes can be used to screen tissue culture for contamination (i.e.screen for the presence of a mixture of different types of cells in aculture).

[0278] Predictive Medicine

[0279] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual.

[0280] Generally, the invention provides, a method of determining if asubject is at risk for a disorder related to a lesion in or themisexpression of a gene which encodes 53014.

[0281] Such disorders include, e.g., a disorder associated with themisexpression of 53014 gene; a disorder of the nervous, cellproliferative/differentiative, skeletal or erythroid system.

[0282] The method includes one or more of the following:

[0283] detecting, in a tissue of the subject, the presence or absence ofa mutation which affects the expression of the 53014 gene, or detectingthe presence or absence of a mutation in a region which controls theexpression of the gene, e.g., a mutation in the 5′ control region;

[0284] detecting, in a tissue of the subject, the presence or absence ofa mutation which alters the structure of the 53014 gene;

[0285] detecting, in a tissue of the subject, the misexpression of the53014 gene, at the mRNA level, e.g., detecting a non-wild type level ofan mRNA;

[0286] detecting, in a tissue of the subject, the misexpression of thegene, at the protein level, e.g., detecting a non-wild type level of a53014 polypeptide.

[0287] In preferred embodiments the method includes: ascertaining theexistence of at least one of: a deletion of one or more nucleotides fromthe 53014 gene; an insertion of one or more nucleotides into the gene, apoint mutation, e.g., a substitution of one or more nucleotides of thegene, a gross chromosomal rearrangement of the gene, e.g., atranslocation, inversion, or deletion.

[0288] For example, detecting the genetic lesion can include: (i)providing a probe/primer including an oligonucleotide containing aregion of nucleotide sequence which hybridizes to a sense or antisensesequence from SEQ ID NO: 1, SEQ ID NO: 4, or naturally occurring mutantsthereof or 5′ or 3′ flanking sequences naturally associated with the53014 gene; (ii) exposing the probe/primer to nucleic acid of thetissue; and detecting, by hybridization, e.g., in situ hybridization, ofthe probe/primer to the nucleic acid, the presence or absence of thegenetic lesion.

[0289] In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of the 53014 gene; the presence of anon-wild type splicing pattern of a messenger RNA transcript of thegene; or a non-wild type level of 53014.

[0290] Methods of the invention can be used prenatally or to determineif a subject's offspring will be at risk for a disorder.

[0291] In preferred embodiments the method includes determining thestructure of a 53014 gene, an abnormal structure being indicative ofrisk for the disorder.

[0292] In preferred embodiments the method includes contacting a samplefrom the subject with an antibody to the 53014 protein or a nucleicacid, which hybridizes specifically with the gene. These and otherembodiments are discussed below.

[0293] Diagnostic and Prognostic Assays

[0294] The presence, level, or absence of 53014 protein or nucleic acidin a biological sample can be evaluated by obtaining a biological samplefrom a test subject and contacting the biological sample with a compoundor an agent capable of detecting 53014 protein or nucleic acid (e.g.,mRNA, genomic DNA) that encodes 53014 protein such that the presence of53014 protein or nucleic acid is detected in the biological sample. Theterm “biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum. The level ofexpression of the 53014 gene can be measured in a number of ways,including, but not limited to: measuring the mRNA encoded by the 53014genes; measuring the amount of protein encoded by the 53014 genes; ormeasuring the activity of the protein encoded by the 53014 genes.

[0295] The level of mRNA corresponding to the 53014 gene in a cell canbe determined both by in situ and by in vitro formats.

[0296] The isolated mRNA can be used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onepreferred diagnostic method for the detection of mRNA levels involvescontacting the isolated mRNA with a nucleic acid molecule (probe) thatcan hybridize to the mRNA encoded by the gene being detected. Thenucleic acid probe can be, for example, a full-length 53014 nucleicacid, such as the nucleic acid of SEQ ID NO: 1, SEQ ID NO: 4, or aportion thereof, such as an oligonucleotide of at least 7, 15, 30, 50,100, 250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to 53014 mRNA or genomic DNA. Othersuitable probes for use in the diagnostic assays are described herein.

[0297] In one format, mRNA (or cDNA) is immobilized on a surface andcontacted with the probes, for example by running the isolated mRNA onan agarose gel and transferring the mRNA from the gel to a membrane,such as nitrocellulose. In an alternative format, the probes areimmobilized on a surface and the mRNA (or cDNA) is contacted with theprobes, for example, in a two-dimensional gene chip array. A skilledartisan can adapt known mRNA detection methods for use in detecting thelevel of mRNA encoded by the 53014 genes.

[0298] The level of mRNA in a sample that is encoded by one of 53014 canbe evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis(1987) U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991)Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al.,(1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al., (1988) Bio/Technology 6:1197), rolling circlereplication (Lizardi et al., U.S. Pat. No. 5,854,033) or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques known in the art. As used herein,amplification primers are defined as being a pair of nucleic acidmolecules that can anneal to 5′ or 3′ regions of a gene (plus and minusstrands, respectively, or vice-versa) and contain a short region inbetween. In general, amplification primers are from about 10 to 30nucleotides in length and flank a region from about 50 to 200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

[0299] For in situ methods, a cell or tissue sample can beprepared/processed and immobilized on a support, typically a glassslide, and then contacted with a probe that can hybridize to mRNA thatencodes the 53014 gene being analyzed.

[0300] In another embodiment, the methods further contacting a controlsample with a compound or agent capable of detecting 53014 mRNA, orgenomic DNA, and comparing the presence of 53014 mRNA or genomic DNA inthe control sample with the presence of 53014 mRNA or genomic DNA in thetest sample.

[0301] A variety of methods can be used to determine the level ofprotein encoded by 53014. In general, these methods include contactingan agent that selectively binds to the protein, such as an antibody witha sample, to evaluate the level of protein in the sample. In a preferredembodiment, the antibody bears a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with a detectablesubstance. Examples of detectable substances are provided herein.

[0302] The detection methods can be used to detect 53014 protein in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of 53014 protein include enzyme linked immunosorbent assays(ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay(EIA), radioimmunoassay (RIA), and Western blot analysis. In vivotechniques for detection of 53014 protein include introducing into asubject a labeled anti-53014 antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

[0303] In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting 53014protein, and comparing the presence of 53014 protein in the controlsample with the presence of 53014 protein in the test sample.

[0304] The invention also includes kits for detecting the presence of53014 in a biological sample. For example, the kit can include acompound or agent capable of detecting 53014 protein or mRNA in abiological sample; and a standard. The compound or agent can be packagedin a suitable container. The kit can further comprise instructions forusing the kit to detect 53014 protein or nucleic acid.

[0305] For antibody-based kits, the kit can include: (1) a firstantibody (e.g., attached to a solid support) which binds to apolypeptide corresponding to a marker of the invention; and, optionally,(2) a second, different antibody which binds to either the polypeptideor the first antibody and is conjugated to a detectable agent.

[0306] For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

[0307] The diagnostic methods described herein can identify subjectshaving, or at risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted 53014 expression or activity. Asused herein, the term “unwanted” includes an unwanted phenomenoninvolved in a biological response such as pain or deregulated cellproliferation.

[0308] In one embodiment, a disease or disorder associated with aberrantor unwanted 53014 expression or activity is identified. A test sample isobtained from a subject and 53014 protein or nucleic acid (e.g., mRNA orgenomic DNA) is evaluated, wherein the level, e.g., the presence orabsence, of 53014 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted 53014 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest, including a biological fluid (e.g., serum), cell sample, ortissue.

[0309] The prognostic assays described herein can be used to determinewhether a subject can be administered an agent (e.g., an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, smallmolecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted 53014 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a cellular proliferative and/ordifferentiative disorder, neurological disorder, hematological disorder,a disorder associated with bone metabolism, an immune, e.g. inflammatorydisorder, or a disorder associated with undesirable or deficientvascularization/angiogenesis.

[0310] The methods of the invention can also be used to detect geneticalterations in a 53014 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation in53014 protein activity or nucleic acid expression, such as a a cellularproliferative and/or differentiative disorder, neurological disorder,hematological disorder, a disorder associated with bone metabolism, animmune, e.g. inflammatory disorder, or a disorder associated withundesirable or deficient vascularization/angiogenesis. In preferredembodiments, the methods include detecting, in a sample from thesubject, the presence or absence of a genetic alteration characterizedby at least one of an alteration affecting the integrity of a geneencoding a 53014-protein, or the mis-expression of the 53014 gene. Forexample, such genetic alterations can be detected by ascertaining theexistence of at least one of 1) a deletion of one or more nucleotidesfrom a 53014 gene; 2) an addition of one or more nucleotides to a 53014gene; 3) a substitution of one or more nucleotides of a 53014 gene, 4) achromosomal rearrangement of a 53014 gene; 5) an alteration in the levelof a messenger RNA transcript of a 53014 gene, 6) aberrant modificationof a 53014 gene, such as of the methylation pattern of the genomic DNA,7) the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a 53014 gene, 8) a non-wild type level of a 53014-protein,9) allelic loss of a 53014 gene, and 10) inappropriatepost-translational modification of a 53014-protein.

[0311] An alteration can be detected without a probe/primer in apolymerase chain reaction, such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR), the latter of whichcan be particularly useful for detecting point mutations in the53014-gene. This method can include the steps of collecting a sample ofcells from a subject, isolating nucleic acid (e.g., genomic, mRNA orboth) from the sample, contacting the nucleic acid sample with one ormore primers which specifically hybridize to a 53014 gene underconditions such that hybridization and amplification of the 53014 gene(if present) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein. Alternatively, other amplificationmethods described herein or known in the art can be used.

[0312] In another embodiment, mutations in a 53014 gene from a samplecell can be identified by detecting alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined, e.g., by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0313] In other embodiments, genetic mutations in 53014 can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, two dimensional arrays, e.g., chip based arrays. Such arraysinclude a plurality of addresses, each of which is positionallydistinguishable from the other. A different probe is located at eachaddress of the plurality. The arrays can have a high density ofaddresses, e.g., can contain hundreds or thousands of oligonucleotidesprobes (Cronin et al. (1996) Human Mutation 7: 244-255; Kozal et al.(1996) Nature Medicine 2: 753-759). For example, genetic mutations in53014 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0314] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the 53014gene and detect mutations by comparing the sequence of the sample 53014with the corresponding wild-type (control) sequence. Automatedsequencing procedures can be utilized when performing the diagnosticassays (Naeve et al. (1995) Biotechniques 19:448-53), includingsequencing by mass spectrometry.

[0315] Other methods for detecting mutations in the 53014 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295).

[0316] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in 53014 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662; U.S. Pat. No. 5,459,039).

[0317] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in 53014 genes. For example, singlestrand conformation polymorphism (SSCP) can be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control 53014 nucleic acids will be denatured and allowed torenature. The secondary structure of single-stranded nucleic acidsvaries according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments can be labeled or detected with labeledprobes. The sensitivity of the assay can be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet 7:5).

[0318] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0319] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. NatlAcad. Sci USA 86:6230).

[0320] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification can be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification can carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification can also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189-93). In such cases, ligation will occur only ifthere is a perfect match at the 3′ end of the 5′ sequence making itpossible to detect the presence of a known mutation at a specific siteby looking for the presence or absence of amplification.

[0321] The methods described herein can be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which can beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga 53014 gene.

[0322] Use of 53014 Molecules as Surrogate Markers

[0323] The 53014 molecules of the invention are also useful as markersof disorders or disease states, as markers for precursors of diseasestates, as markers for predisposition of disease states, as markers ofdrug activity, or as markers of the pharmacogenomic profile of asubject. Using the methods described herein, the presence, absenceand/or quantity of the 53014 molecules of the invention can be detected,and can be correlated with one or more biological states in vivo. Forexample, the 53014 molecules of the invention can serve as surrogatemarkers for one or more disorders or disease states or for conditionsleading up to disease states. As used herein, a “surrogate marker” is anobjective biochemical marker which correlates with the absence orpresence of a disease or disorder, or with the progression of a diseaseor disorder (e.g., with the presence or absence of a tumor). Thepresence or quantity of such markers is independent of the disease.Therefore, these markers can serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies (e.g., early stage tumors), or when an assessmentof disease progression is desired before a potentially dangerousclinical endpoint is reached (e.g., an assessment of cardiovasculardisease can be made using cholesterol levels as a surrogate marker, andan analysis of HIV infection can be made using HIV RNA levels as asurrogate marker, well in advance of the undesirable clinical outcomesof myocardial infarction or fully-developed AIDS). Examples of the useof surrogate markers in the art include: Koomen et al. (2000) J. Mass.Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[0324] The 53014 molecules of the invention are also useful aspharmacodynamic markers. As used herein, a “pharmacodynamic marker” isan objective biochemical marker which correlates specifically with drugeffects. The presence or quantity of a pharmacodynamic marker is notrelated to the disease state or disorder for which the drug is beingadministered; therefore, the presence or quantity of the marker isindicative of the presence or activity of the drug in a subject. Forexample, a pharmacodynamic marker can be indicative of the concentrationof the drug in a biological tissue, in that the marker is eitherexpressed or transcribed or not expressed or transcribed in that tissuein relationship to the level of the drug. In this fashion, thedistribution or uptake of the drug can be monitored by thepharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker can be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug can besufficient to activate multiple rounds of marker (e.g., a 53014 marker)transcription or expression, the amplified marker can be in a quantitywhich is more readily detectable than the drug itself. Also, the markercan be more easily detected due to the nature of the marker itself; forexample, using the methods described herein, anti-53014 antibodies canbe employed in an immune-based detection system for a 53014 proteinmarker, or 53014-specific radiolabeled probes can be used to detect a53014 mRNA marker. Furthermore, the use of a pharmacodynamic marker canoffer mechanism-based prediction of risk due to drug treatment beyondthe range of possible direct observations. Examples of the use ofpharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No.6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238;Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; andNicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[0325] The 53014 molecules of the invention are also useful aspharmacogenomic markers. As used herein, a “pharmacogenomic marker” isan objective biochemical marker which correlates with a specificclinical drug response or susceptibility in a subject (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity ofthe pharmacogenomic marker is related to the predicted response of thesubject to a specific drug or class of drugs prior to administration ofthe drug. By assessing the presence or quantity of one or morepharmacogenomic markers in a subject, a drug therapy which is mostappropriate for the subject, or which is predicted to have a greaterdegree of success, can be selected. For example, based on the presenceor quantity of RNA, or protein (e.g., 53014 protein or RNA) for specifictumor markers in a subject, a drug or course of treatment can beselected that is optimized for the treatment of the specific tumorlikely to be present in the subject. Similarly, the presence or absenceof a specific sequence mutation in 53014 DNA can correlate with a 53014drug response. The use of pharmacogenomic markers therefore permits theapplication of the most appropriate treatment for each subject withouthaving to administer the therapy.

[0326] Pharmaceutical Compositions

[0327] The nucleic acid and polypeptides, fragments thereof, as well asanti-53014 antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositions. Suchcompositions typically include the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” includes solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

[0328] A pharmaceutical composition is formulated to be compatible withits intended route of administration. Examples of routes ofadministration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral, transdermal (e.g. topical), transmucosal (e.g.,inhalation of aerosol or absorption of eye drop), and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0329] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0330] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0331] Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0332] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0333] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0334] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0335] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0336] It is advantageous to formulate oral or parenteral compositionsin dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

[0337] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit high therapeutic indices arepreferred. While compounds that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0338] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

[0339] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody, unconjugated or conjugated asdescribed herein, can include a single treatment or, preferably, caninclude a series of treatments.

[0340] For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

[0341] The present invention encompasses agents which modulateexpression or activity. An agent can, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e.,. includingheteroorganic and organometallic compounds) having a molecular weightless than about 10,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 5,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 1,000grams per mole, organic or inorganic compounds having a molecular weightless than about 500 grams per mole, and salts, esters, and otherpharmaceutically acceptable forms of such compounds.

[0342] Exemplary doses include milligram or microgram amounts of thesmall molecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher can, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0343] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0344] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0345] Methods of Treatment:

[0346] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant or unwanted53014 expression or activity. As used herein, the term “treatment” isdefined as the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

[0347] With regards to both prophylactic and therapeutic methods oftreatment, such treatments can be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Phannacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the 53014 molecules ofthe present invention or 53014 modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and not to provide this treatment topatients who will experience toxic drug-related side effects.

[0348] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant orunwanted 53014 expression or activity, by administering to the subject a53014 or an agent which modulates 53014 expression or at least one 53014activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted 53014 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe 53014 aberrance, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of53014 aberrance, for example, a 53014, 53014 agonist or 53014 antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

[0349] It is possible that some 53014 disorders can be caused, at leastin part, by an abnormal level of gene product, or by the presence of agene product exhibiting abnormal activity. As such, the reduction in thelevel and/or activity of such gene products would bring about theamelioration of disorder symptoms.

[0350] The 53014 molecules can act as novel diagnostic targets andtherapeutic agents for controlling one or more of cellular proliferativeand/or differentiative disorders, neurological disorders, disordersassociated with bone metabolism, erythroid cell associated disorders,immune e.g., inflammatory, disorders, disorders associated withundesirable or deficient vascularization/angiogenesis, disordersassociated with undesirable extracellular matrix accumulation, e.g.,characterized by fibrosis or a scar, liver disorders, or viral diseases,all of which are described above. The molecules of the invention alsocan act as novel diagnostic targets and therapeutic agents forcontrolling one or more of cardiovascular disorders, endothelial celldisorders, pain disorders and metabolic disorders.

[0351] As used herein, disorders involving the heart, or “cardiovasculardisease” or a “cardiovascular disorder” includes a disease or disorderwhich affects the cardiovascular system, e.g., the heart, the bloodvessels, and/or the blood. A cardiovascular disorder can be caused by animbalance in arterial pressure, a malfunction of the heart, or anocclusion of a blood vessel, e.g., by a thrombus. A cardiovasculardisorder includes, but is not limited to disorders such asarteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemiareperfusion injury, restenosis, arterial inflammation, vascular wallremodeling, ventricular remodeling, rapid ventricular pacing, coronarymicroembolism, tachycardia, bradycardia, pressure overload, aorticbending, coronary artery ligation, vascular heart disease, valvulardisease, including but not limited to, valvular degeneration caused bycalcification, rheumatic heart disease, endocarditis, or complicationsof artificial valves; atrial fibrillation, long-QT syndrome, congestiveheart failure, sinus node dysfunction, angina, heart failure,hypertension, atrial fibrillation, atrial flutter, pericardial disease,including but not limited to, pericardial effusion and pericarditis;cardiomyopathies, e.g., dilated cardiomyopathy or idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, ischemic disease, arrhythmia, sudden cardiac death, andcardiovascular developmental disorders (e.g., arteriovenousmalformations, arteriovenous fistulae, raynaud's syndrome, neurogenicthoracic outlet syndrome, causalgia/reflex sympathetic dystrophy,hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrialseptal defects, atrioventricular canal, coarctation of the aorta,ebsteins anomaly, hypoplastic left heart syndrome, interruption of theaortic arch, mitral valve prolapse, ductus arteriosus, patent foramenovale, partial anomalous pulmonary venous return, pulmonary atresia withventricular septal defect, pulmonary atresia without ventricular septaldefect, persistance of the fetal circulation, pulmonary valve stenosis,single ventricle, total anomalous pulmonary venous return, transpositionof the great vessels, tricuspid atresia, truncus arteriosus, ventricularseptal defects). A cardiovascular disease or disorder also can includean endothelial cell disorder.

[0352] As used herein, an “endothelial cell disorder” includes adisorder characterized by aberrant, unregulated, or unwanted endothelialcell activity, e.g., proliferation, migration, angiogenesis, orvascularization; or aberrant expression of cell surface adhesionmolecules or genes associated with angiogenesis, e.g., TIE-2, FLT andFLK. Endothelial cell disorders include tumorigenesis, tumor metastasis,psoriasis, diabetic retinopathy, endometriosis, Grave's disease,ischemic disease (e.g., atherosclerosis), and chronic inflammatorydiseases (e.g., rheumatoid arthritis).

[0353] Additionally, 53014 can play an important role in the regulationof metabolism or pain disorders. Diseases of metabolic imbalanceinclude, but are not limited to, obesity, anorexia nervosa, cachexia,lipid disorders, and diabetes. Examples of pain disorders include, butare not limited to, pain response elicited during various forms oftissue injury, e.g., inflammation, infection, and ischemia, usuallyreferred to as hyperalgesia (described in, for example, Fields, H. L.(1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletaldisorders, e.g., joint pain; tooth pain; headaches; pain associated withsurgery; pain related to irritable bowel syndrome; or chest pain.

[0354] As discussed, successful treatment of 53014 disorders can bebrought about by techniques that serve to inhibit the expression oractivity of target gene products. For example, compounds, e.g., an agentidentified using an assays described above, that proves to exhibitnegative modulatory activity, can be used in accordance with theinvention to prevent and/or ameliorate symptoms of 53014 disorders. Suchmolecules can include, but are not limited to peptides, phosphopeptides,small organic or inorganic molecules, or antibodies (including, forexample, polyclonal, monoclonal, humanized, human, anti-idiotypic,chimeric or single chain antibodies, and Fab, F(ab′)₂ and Fab expressionlibrary fragments, scFV molecules, and epitope-binding fragmentsthereof).

[0355] Further, antisense and ribozyme molecules that inhibit expressionof the target gene can also be used in accordance with the invention toreduce the level of target gene expression, thus effectively reducingthe level of target gene activity. Still further, triple helix moleculescan be utilized in reducing the level of target gene activity.Antisense, ribozyme and triple helix molecules are discussed above.

[0356] It is possible that the use of antisense, ribozyme, and/or triplehelix molecules to reduce or inhibit mutant gene expression can alsoreduce or inhibit the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal target gene alleles,such that the concentration of normal target gene product present can belower than is necessary for a normal phenotype. In such cases, nucleicacid molecules that encode and express target gene polypeptidesexhibiting normal target gene activity can be introduced into cells viagene therapy method. Alternatively, in instances in that the target geneencodes an extracellular protein, it can be preferable to co-administernormal target gene protein into the cell or tissue in order to maintainthe requisite level of cellular or tissue target gene activity.

[0357] Another method by which nucleic acid molecules can be utilized intreating or preventing a disease characterized by 53014 expression isthrough the use of aptamer molecules specific for 53014 protein.Aptamers are nucleic acid molecules having a tertiary structure whichpermits them to specifically or selectively bind to protein ligands(see, e.g., Osborne et al. (1997) Curr. Opin. Chem Biol. 1:5-9; andPatel (1997) Curr Opin Chem Biol 1:32-46). Since nucleic acid moleculescan in many cases be more conveniently introduced into target cells thantherapeutic protein molecules can be, aptamers offer a method by which53014 protein activity can be specifically decreased without theintroduction of drugs or other molecules which can have pluripotenteffects.

[0358] Antibodies can be generated that are both specific for targetgene product and that reduce target gene product activity. Suchantibodies can, therefore, by administered in instances whereby negativemodulatory techniques are appropriate for the treatment of 53014disorders. For a description of antibodies, see the Antibody sectionabove.

[0359] In circumstances wherein injection of an animal or a humansubject with a 53014 protein or epitope for stimulating antibodyproduction is harmful to the subject, it is possible to generate animmune response against 53014 through the use of anti-idiotypicantibodies (see, for example, Herlyn (1999) Ann Med 31:66-78; andBhattacharya-Chatterjee and Foon (1998) Cancer Treat Res. 94:51-68). Ifan anti-idiotypic antibody is introduced into a mammal or human subject,it should stimulate the production of anti-anti-idiotypic antibodies,which should be specific to the 53014 protein. Vaccines directed to adisease characterized by 53014 expression can also be generated in thisfashion.

[0360] In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies can be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.(1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0361] The identified compounds that inhibit target gene expression,synthesis and/or activity can be administered to a patient attherapeutically effective doses to prevent, treat or ameliorate 53014disorders. A therapeutically effective dose refers to that amount of thecompound sufficient to result in amelioration of symptoms of thedisorders. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures as described above.

[0362] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

[0363] Another example of determination of effective dose for anindividual is the ability to directly assay levels of “free” and “bound”compound in the serum of the test subject. Such assays can utilizeantibody mimics and/or “biosensors” that have been created throughmolecular imprinting techniques. The compound which is able to modulate53014 activity is used as a template, or “imprinting molecule”, tospatially organize polymerizable monomers prior to their polymerizationwith catalytic reagents. The subsequent removal of the imprintedmolecule leaves a polymer matrix which contains a repeated “negativeimage” of the compound and is able to selectively rebind the moleculeunder biological assay conditions. A detailed review of this techniquecan be seen in Ansell et al (1996) Current Opinion in Biotechnology7:89-94 and in Shea (1994) Trends in Polymer Science 2:166-173. Such“imprinted” affinity matrixes are amenable to ligand-binding assays,whereby the immobilized monoclonal antibody component is replaced by anappropriately imprinted matrix. An example of the use of such matrixesin this way can be seen in Vlatakis et al (1993) Nature 361:645-647.Through the use of isotope-labeling, the “free” concentration ofcompound which modulates the expression or activity of 53014 can bereadily monitored and used in calculations of IC₅₀.

[0364] Such “imprinted” affinity matrixes can also be designed toinclude fluorescent groups whose photon-emitting properties measurablychange upon local and selective binding of target compound. Thesechanges can be readily assayed in real time using appropriate fiberopticdevices, in turn allowing the dose in a test subject to be quicklyoptimized based on its individual IC₅₀. An rudimentary example of such a“biosensor” is discussed in Kriz et al (1995) Analytical Chemistry67:2142-2144.

[0365] Another aspect of the invention pertains to methods of modulating53014 expression or activity for therapeutic purposes. Accordingly, inan exemplary embodiment, the modulatory method of the invention involvescontacting a cell with a 53014 or agent that modulates one or more ofthe activities of 53014 protein activity associated with the cell. Anagent that modulates 53014 protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a 53014 protein (e.g., a 53014 substrate orreceptor), a 53014 antibody, a 53014 agonist or antagonist, apeptidomimetic of a 53014 agonist or antagonist, or other smallmolecule.

[0366] In one embodiment, the agent stimulates one or 53014 activities.Examples of such stimulatory agents include active 53014 protein and anucleic acid molecule encoding 53014. In another embodiment, the agentinhibits one or more 53014 activities. Examples of such inhibitoryagents include antisense 53014 nucleic acid molecules, anti-53014antibodies, and 53014 inhibitors. These modulatory methods can beperformed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a 53014 protein ornucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g., upregulates or down regulates) 53014 expression or activity. In anotherembodiment, the method involves administering a 53014 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted 53014 expression or activity.

[0367] Stimulation of 53014 activity is desirable in situations in which53014 is abnormally downregulated and/or in which increased 53014activity is likely to have a beneficial effect. For example, stimulationof 53014 activity is desirable in situations in which a 53014 isdownregulated and/or in which increased 53014 activity is likely to havea beneficial effect. Likewise, inhibition of 53014 activity is desirablein situations in which 53014 is abnormally upregulated and/or in whichdecreased 53014 activity is likely to have a beneficial effect.

[0368] Pharmacogenomics

[0369] The 53014 molecules of the present invention, as well as agents,or modulators which have a stimulatory or inhibitory effect on 53014activity (e.g., 53014 gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) 53014-associated disorders (e.g.,aberrant or deficient metalloprotease, e.g. reprolysin (M12B) (e.g.ADAMTS) function or expression) associated with aberrant or unwanted53014 activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) can beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician can consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer a53014 molecule or 53014 modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a 53014 molecule or 53014modulator.

[0370] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum et al.(1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder et al.(1997) Clin. Chem. 43:254-266. In general, two types of pharmacogeneticconditions can be differentiated. Genetic conditions transmitted as asingle factor altering the way drugs act on the body (altered drugaction) or genetic conditions transmitted as single factors altering theway the body acts on drugs (altered drug metabolism). Thesepharmacogenetic conditions can occur either as rare genetic defects oras naturally-occurring polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinical complication is haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

[0371] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP can occur once per every 1000 bases of DNA. ASNP can be involved in a disease process, however, the vast majority cannot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that can becommon among such genetically similar individuals.

[0372] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drug's target is known (e.g., a53014 protein of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

[0373] Alternatively, a method termed the “gene expression profiling”,can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a drug (e.g., a53014 molecule or 53014 modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

[0374] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment of anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with a53014 molecule or 53014 modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

[0375] The present invention further provides methods for identifyingnew agents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the 53014 genes of the present invention, wherein theseproducts can be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by the 53014genes of the present invention can be used as a basis for identifyingagents for overcoming agent resistance. By blocking the activity of oneor more of the resistance proteins, target cells, e.g., human cells,will become sensitive to treatment with an agent to which the unmodifiedtarget cells were resistant.

[0376] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a 53014 protein can be applied in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase 53014 gene expression,protein levels, or upregulate 53014 activity, can be monitored inclinical trials of subjects exhibiting decreased 53014 gene expression,protein levels, or downregulated 53014 activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decrease53014 gene expression, protein levels, or downregulate 53014 activity,can be monitored in clinical trials of subjects exhibiting increased53014 gene expression, protein levels, or upregulated 53014 activity. Insuch clinical trials, the expression or activity of a 53014 gene, andpreferably, other genes that have been implicated in, for example, ametalloprotease-associated or another 53014-associated disorder can beused as a “read out” or markers of the phenotype of a particular cell.

[0377] Other Embodiments

[0378] In another aspect, the invention features a method of analyzing aplurality of capture probes. The method is useful, e.g., to analyze geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence, wherein the capture probes are from acell or subject which expresses 53014 or from a cell or subject in whicha 53014 mediated response has been elicited; contacting the array with a53014 nucleic acid (preferably purified), a 53014 polypeptide(preferably purified), or an anti-53014 antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by a signal generated from a label attached to the53014 nucleic acid, polypeptide, or antibody.

[0379] The capture probes can be a set of nucleic acids from a selectedsample, e.g., a sample of nucleic acids derived from a control ornon-stimulated tissue or cell.

[0380] The method can include contacting the 53014 nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

[0381] The plurality of capture probes can be a plurality of nucleicacid probes each of which specifically hybridizes, with an allele of53014. Such methods can be used to diagnose a subject, e.g., to evaluaterisk for a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder.

[0382] The method can be used to detect SNPs, as described above.

[0383] In another aspect, the invention features, a method of analyzing53014, e.g., analyzing structure, function, or relatedness to othernucleic acid or amino acid sequences. The method includes: providing a53014 nucleic acid or amino acid sequence; comparing the 53014 sequencewith one or more preferably a plurality of sequences from a collectionof sequences, e.g., a nucleic acid or protein sequence database; tothereby analyze 53014.

[0384] The method can include evaluating the sequence identity between a53014 sequence and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.Preferred databases include GenBank™ and SwissProt.

[0385] In another aspect, the invention features, a set ofoligonucleotides, useful, e.g., for identifying SNP's, or identifyingspecific alleles of 53014. The set includes a plurality ofoligonucleotides, each of which has a different nucleotide at aninterrogation position, e.g., an SNP or the site of a mutation. In apreferred embodiment, the oligonucleotides of the plurality identical insequence with one another (except for differences in length). Theoligonucleotides can be provided with differential labels, such that anoligonucleotide which hybridizes to one allele provides a signal that isdistinguishable from an oligonucleotides which hybridizes to a secondallele.

[0386] The sequences of 53014 molecules are provided in a variety ofmediums to facilitate use thereof. A sequence can be provided as amanufacture, other than an isolated nucleic acid or amino acid molecule,which contains a 53014 molecule. Such a manufacture can provide anucleotide or amino acid sequence, e.g., an open reading frame, in aform which allows examination of the manufacture using means notdirectly applicable to examining the nucleotide or amino acid sequences,or a subset thereof, as they exist in nature or in purified form.

[0387] A 53014 nucleotide or amino acid sequence can be recorded oncomputer readable media. As used herein, “computer readable media”refers to any medium that can be read and accessed directly by acomputer. Such media include, but are not limited to: magnetic storagemedia, such as floppy discs, hard disc storage medium, and magnetictape; optical storage media such as compact disc and CD-ROM; electricalstorage media such as RAM, ROM, EPROM, EEPROM, and the like; and generalhard disks and hybrids of these categories such as magnetic/opticalstorage media. The medium is adapted or configured for having thereon53014 sequence information of the present invention.

[0388] As used herein, the term “electronic apparatus” is intended toinclude any suitable computing or processing apparatus of other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phones, pagers, and the like; and local and distributedprocessing systems.

[0389] As used herein, “recorded” refers to a process for storing orencoding information on the electronic apparatus readable medium. Thoseskilled in the art can readily adopt any of the presently known methodsfor recording information on known media to generate manufacturescomprising the 53014 sequence information.

[0390] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona 53014 nucleotide or amino acid sequence of the present invention. Thechoice of the data storage structure will generally be based on themeans chosen to access the stored information. In addition, a variety ofdata processor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g., text file or database) in order toobtain computer readable medium having recorded thereon the nucleotidesequence information of the present invention.

[0391] By providing the 53014 nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. A search is used to identify fragments or regions ofthe sequences of the invention which match a particular target sequenceor target motif.

[0392] The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a metalloprotease-associated or another 53014-associated disease ordisorder or a pre-disposition to a metalloprotease-associated or another53014-associated disease or disorder, wherein the method comprises thesteps of determining 53014 sequence information associated with thesubject and based on the 53014 sequence information, determining whetherthe subject has a metalloprotease-associated or another 53014-associateddisease or disorder and/or recommending a particular treatment for thedisease, disorder, or pre-disease condition.

[0393] The present invention further provides in an electronic systemand/or in a network, a method for determining whether a subject has ametalloprotease-associated or another 53014-associated disease ordisorder or a pre-disposition to a disease associated with 53014,wherein the method comprises the steps of determining 53014 sequenceinformation associated with the subject, and based on the 53014 sequenceinformation, determining whether the subject has ametalloprotease-associated or another 53014-associated disease ordisorder or a pre-disposition to a metalloprotease-associated or another53014-associated disease or disorder, and/or recommending a particulartreatment for the disease, disorder, or pre-disease condition. Themethod may further comprise the step of receiving phenotypic informationassociated with the subject and/or acquiring from a network phenotypicinformation associated with the subject.

[0394] The present invention also provides in a network, a method fordetermining whether a subject has a metalloprotease-associated oranother 53014-associated disease or disorder or a pre-disposition to ametalloprotease-associated or another 53014-associated disease ordisorder, said method comprising the steps of receiving 53014 sequenceinformation from the subject and/or information related thereto,receiving phenotypic information associated with the subject, acquiringinformation from the network corresponding to 53014 and/or correspondingto a metalloprotease-associated or another 53014-associated disease ordisorder, and based on one or more of the phenotypic information, the53014 information (e.g., sequence information and/or information relatedthereto), and the acquired information, determining whether the subjecthas a metalloprotease-associated or another 53014-associated disease ordisorder or a pre-disposition to a metalloprotease-associated or another53014-associated disease or disorder. The method may further comprisethe step of recommending a particular treatment for the disease,disorder, or pre-disease condition.

[0395] The present invention also provides a business method fordetermining whether a subject has a metalloprotease-associated oranother 53014-associated disease or disorder or a pre-disposition to ametalloprotease-associated or another 53014-associated disease ordisorder, said method comprising the steps of receiving informationrelated to 53014 (e.g., sequence information and/or information relatedthereto), receiving phenotypic information associated with the subject,acquiring information from the network related to 53014 and/or relatedto a metalloprotease-associated or another 53014-associated disease ordisorder, and based on one or more of the phenotypic information, the53014 information, and the acquired information, determining whether thesubject has a metalloprotease-associated or another 53014-associateddisease or disorder or a pre-disposition to a metalloprotease-associatedor another 53014-associated disease or disorder. The method may furthercomprise the step of recommending a particular treatment for thedisease, disorder, or pre-disease condition.

[0396] The invention also includes an array comprising a 53014 sequenceof the present invention. The array can be used to assay expression ofone or more genes in the array. In one embodiment, the array can be usedto assay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be 53014. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

[0397] In addition to such qualitative information, the invention allowsthe quantitation of gene expression. Thus, not only tissue specificity,but also the level of expression of a battery of genes in the tissue ifascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionin that tissue. Thus, one tissue can be perturbed and the effect on geneexpression in a second tissue can be determined. In this context, theeffect of one cell type on another cell type in response to a biologicalstimulus can be determined. In this context, the effect of one cell typeon another cell type in response to a biological stimulus can bedetermined. Such a determination is useful, for example, to know theeffect of cell-cell interaction at the level of gene expression. If anagent is administered therapeutically to treat one cell type but has anundesirable effect on another cell type, the invention provides an assayto determine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

[0398] In another embodiment, the array can be used to monitor the timecourse of expression of one or more genes in the array. This can occurin various biological contexts, as disclosed herein, for exampledevelopment of a metalloprotease-associated or another 53014-associateddisease or disorder, progression of metalloprotease-associated oranother 53014-associated disease or disorder, and processes, such acellular transformation associated with the metalloprotease-associatedor another 53014-associated disease or disorder.

[0399] The array is also useful for ascertaining the effect of theexpression of a gene on the expression of other genes in the same cellor in different cells (e.g., acertaining the effect of 53014 expressionon the expression of other genes). This provides, for example, for aselection of alternate molecular targets for therapeutic intervention ifthe ultimate or downstream target cannot be regulated.

[0400] The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including 53014) that could serve asa molecular target for diagnosis or therapeutic intervention.

[0401] As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. Typical sequence lengths of a targetsequence are from about 10 to 100 amino acids or from about 30 to 300nucleotide residues. However, it is well recognized that commerciallyimportant fragments, such as sequence fragments involved in geneexpression and protein processing, may be of shorter length.

[0402] Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium for analysis and comparison to other sequences. A variety ofknown algorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but are not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBI).

[0403] Thus, the invention features a method of making a computerreadable record of a sequence of a 53014 sequence which includesrecording the sequence on a computer readable matrix. In a preferredembodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

[0404] In another aspect, the invention features a method of analyzing asequence. The method includes: providing a 53014 sequence, or record, incomputer readable form; comparing a second sequence to the 53014sequence; thereby analyzing a sequence. Comparison can include comparingto sequences for sequence identity or determining if one sequence isincluded within the other, e.g., determining if the 53014 sequenceincludes a sequence being compared. In a preferred embodiment the 53014or second sequence is stored on a first computer, e.g., at a first siteand the comparison is performed, read, or recorded on a second computer,e.g., at a second site. E.g., the 53014 or second sequence can be storedin a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer. In apreferred embodiment the record includes one or more of the following:identification of an ORF; identification of a domain, region, or site;identification of the start of transcription; identification of thetranscription terminator; the full length amino acid sequence of theprotein, or a mature form thereof; the 5′ end of the translated region.

[0405] This invention is further illustrated by the followingexemplification, which should not be construed as limiting.

EXEMPLIFICATION

[0406] Gene Expression Analysis

[0407] Total RNA was prepared from various human tissues by a singlestep extraction method using RNA STAT-60 according to the manufacturer'sinstructions (TelTest, Inc). Each RNA preparation was treated with DNaseI (Ambion) at 37° C. for 1 hour. DNAse I treatment was determined to becomplete if the sample required at least 38 PCR amplification cycles toreach a threshold level of fluorescence using β-2 microglobulin as aninternal amplicon reference. The integrity of the RNA samples followingDNase I treatment was confirmed by agarose gel electrophoresis andethidium bromide staining. After phenol extraction cDNA was preparedfrom the sample using the SUPERSCRIPT™ Choice System following themanufacturer's instructions (GibcoBRL). A negative control of RNAwithout reverse transcriptase was mock reverse transcribed for each RNAsample.

[0408] Human 53014 expression was measured by TaqMan® quantitative PCR(Perkin Elmer Applied Biosystems) in cDNA prepared from a variety ofnormal and diseased (e.g., cancerous) human tissues or cell lines.

[0409] Probes were designed by PrimerExpress software (PE Biosystems)based on the sequence of the human 53014 gene. Each human 53014 geneprobe was labeled using FAM (6-carboxyfluorescein), and theβ2-microglobulin reference probe was labeled with a differentfluorescent dye, VIC. The differential labeling of the target gene andinternal reference gene thus enabled measurement in same well. Forwardand reverse primers and the probes for both β2-microglobulin and targetgene were added to the TaqMan® Universal PCR Master Mix (PE AppliedBiosystems). Although the final concentration of primer and probe couldvary, each was internally consistent within a given experiment. Atypical experiment contained 200 nM of forward and reverse primers plus100 nM probe for β-2 microglobulin and 600 nM forward and reverseprimers plus 200 nM probe for the target gene. TaqMan matrix experimentswere carried out on an ABI PRISM 7700 Sequence Detection System (PEApplied Biosystems). The thermal cycler conditions were as follows: holdfor 2 min at 50° C. and 10 min at 95° C., followed by two-step PCR for40 cycles of 95° C. for 15 sec followed by 60° C. for 1 min.

[0410] The following method was used to quantitatively calculate human53014 gene expression in the various tissues relative to β-2microglobulin expression in the same tissue. The threshold cycle (Ct)value is defined as the cycle at which a statistically significantincrease in fluorescence is detected. A lower Ct value is indicative ofa higher mRNA concentration. The Ct value of the human 53014 gene isnormalized by subtracting the Ct value of the β-2 microglobulin gene toobtain a _(Δ)Ct value using the following formula:_(Δ)Ct=Ct_(human 59914 and 59921)−Ct_(β-2 microglobulin). Expression isthen calibrated against a cDNA sample showing a comparatively low levelof expression of the human 53014 gene. The _(Δ)Ct value for thecalibrator sample is then subtracted from _(Δ)Ct for each tissue sampleaccording to the following formula:_(ΔΔ)Ct=_(Δ)Ct-_(Sample)−_(Δ)Ct-_(calibrator). Relative expression isthen calculated using the arithmetic formula given by 2^(−ΔΔct).Expression of the target human 53014 gene in each of the tissues testedis then graphically represented as discussed in more detail below.

[0411] The results indicate high levels of 53014 mRNA expression inbrain cortex, spinal cord, hypothalamus, colon adenocarcinoma, lungtumor, ovary tumor, primary osteoblasts, erythroid cells and the K562erythroid cell line. Small amounts of 53014 mRNA expression also werefound in dorsal root ganglion, breast tumor, hemangioma, normal heart,chngestive heart failure heart, skeletal muscle, normal small intestine,normal breast, normal ovary, prostate adenocarcinoma, normal colon, andnormal tonsil. Trace amounts of 53014 mRNA expression were found incoronary smooth muscle cells, kidney, normal liver, normal femalebladder, normal adrenal gland, nerve, benign prostatic hypertrophyprostate, normal lung, inflammatory bowel disease colon, synovium,normal lymph node, normal spleen, macrophages, erythroid progenitors andactivated peripheral blood monocytes.

[0412] The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference.

[0413] Equivalents

[0414] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein.

1 11 1 5701 DNA Homo sapiens CDS (373)...(4044) 1 gccccgccca gtgtgcccacccccggcccg tcccgccgcc ccgtcgcccg gctcccgcgg 60 agtcccggag cgtcacwgssccacgcgcgc gaccgaccag ccggcagttg gcaccgcgtg 120 cggctggcgc gtcagtggccccgctctcca gccggcagcc tcgcgcgccc ggagccagcg 180 gtccaggcgg cggcgccgcgcaggggaccc ggagcaggcg ggagggaagc agctaggcgg 240 ggaggcggct gaggcggcagcggcggcagc cagccggtgc tccgacagcc cggggcgcac 300 cctagcctcg cagccctcagcctgggactt ggggctcggg cgcttgccca gcccgcgtcc 360 cagcgcggcc ac atg gctcca ctc cgc gcg ctg ctg tcc tac ctg ctg cct 411 Met Ala Pro Leu Arg AlaLeu Leu Ser Tyr Leu Leu Pro 1 5 10 ttg cac tgt gcg ctc tgc gcc gcc gcgggc agc cgg acc cca gag ctg 459 Leu His Cys Ala Leu Cys Ala Ala Ala GlySer Arg Thr Pro Glu Leu 15 20 25 cac ctc tct gga aag ctc agt gac tat ggtgtg aca gtg ccc tgc agc 507 His Leu Ser Gly Lys Leu Ser Asp Tyr Gly ValThr Val Pro Cys Ser 30 35 40 45 aca gac ttt cgg gga cgc ttc ctc tcc cacgtg gtg tct ggc cca gca 555 Thr Asp Phe Arg Gly Arg Phe Leu Ser His ValVal Ser Gly Pro Ala 50 55 60 gca gcc tct gca ggg agc atg gta gtg gac acgcca ccc aca cta cca 603 Ala Ala Ser Ala Gly Ser Met Val Val Asp Thr ProPro Thr Leu Pro 65 70 75 cga cac tcc agt cac ctc cgg gtg gct cgc agc cctctg cac cca gga 651 Arg His Ser Ser His Leu Arg Val Ala Arg Ser Pro LeuHis Pro Gly 80 85 90 ggg acc ctg tgg cct ggc agg gtg ggg cgc cac tcc ctctac ttc aat 699 Gly Thr Leu Trp Pro Gly Arg Val Gly Arg His Ser Leu TyrPhe Asn 95 100 105 gtc act gtt ttc ggg aag gaa ctg cac ttg cgc ctg cggccc aat cgg 747 Val Thr Val Phe Gly Lys Glu Leu His Leu Arg Leu Arg ProAsn Arg 110 115 120 125 agg ttg gta gtg cca gga tcc tca gtg gag tgg caggag gat ttt cgg 795 Arg Leu Val Val Pro Gly Ser Ser Val Glu Trp Gln GluAsp Phe Arg 130 135 140 gag ctg ttc cgg cag ccc tta cgg cag gag tgt gtgtac act gga ggt 843 Glu Leu Phe Arg Gln Pro Leu Arg Gln Glu Cys Val TyrThr Gly Gly 145 150 155 gtc act gga atg cct ggg gca gct gtt gcc atc agcaac tgt gac gga 891 Val Thr Gly Met Pro Gly Ala Ala Val Ala Ile Ser AsnCys Asp Gly 160 165 170 ttg gcg ggc ctc atc cgc aca gac agc acc gac ttcttc att gag cct 939 Leu Ala Gly Leu Ile Arg Thr Asp Ser Thr Asp Phe PheIle Glu Pro 175 180 185 ctg gag cgg ggc cag cag gag aag gag gcc agc gggagg aca cat gtg 987 Leu Glu Arg Gly Gln Gln Glu Lys Glu Ala Ser Gly ArgThr His Val 190 195 200 205 gtg tac cgc cgg gag gcc gtc cag cag gag tgggca gaa cct gac ggg 1035 Val Tyr Arg Arg Glu Ala Val Gln Gln Glu Trp AlaGlu Pro Asp Gly 210 215 220 gac ctg cac aat gaa gcc ttt ggc ctg gga gacctt ccc aac ctg ctg 1083 Asp Leu His Asn Glu Ala Phe Gly Leu Gly Asp LeuPro Asn Leu Leu 225 230 235 ggc ctg gtg ggg gac cag ctg ggc gac aca gagcgg aag cgg cgg cat 1131 Gly Leu Val Gly Asp Gln Leu Gly Asp Thr Glu ArgLys Arg Arg His 240 245 250 gcc aag cca ggc agc tac agc atc gag gtg ctgctg gtg gtg gac gac 1179 Ala Lys Pro Gly Ser Tyr Ser Ile Glu Val Leu LeuVal Val Asp Asp 255 260 265 tcg gtg gtt cgc ttc cat ggc aag gag cat gtgcag aac tat gtc ctc 1227 Ser Val Val Arg Phe His Gly Lys Glu His Val GlnAsn Tyr Val Leu 270 275 280 285 acc ctc atg aat atc gta gat gag att taccac gat gag tcc ctg ggg 1275 Thr Leu Met Asn Ile Val Asp Glu Ile Tyr HisAsp Glu Ser Leu Gly 290 295 300 gtt cat ata aat att gcc ctc gtc cgc ttgatc atg gtt ggc tac cga 1323 Val His Ile Asn Ile Ala Leu Val Arg Leu IleMet Val Gly Tyr Arg 305 310 315 cag tcc ctg agc ctg atc gag cgc ggg aacccc tca cgc agc ctg gag 1371 Gln Ser Leu Ser Leu Ile Glu Arg Gly Asn ProSer Arg Ser Leu Glu 320 325 330 cag gtg tgt cgc tgg gca cac tcc cag cagcgc cag gac ccc agc cac 1419 Gln Val Cys Arg Trp Ala His Ser Gln Gln ArgGln Asp Pro Ser His 335 340 345 gct gag cac cat gac cac gtt gtg ttc ctcacc cgg cag gac ttt ggg 1467 Ala Glu His His Asp His Val Val Phe Leu ThrArg Gln Asp Phe Gly 350 355 360 365 ccc tca ggg tat gca ccc gtc act ggcatg tgt cac ccc ctg agg agc 1515 Pro Ser Gly Tyr Ala Pro Val Thr Gly MetCys His Pro Leu Arg Ser 370 375 380 tgt gcc ctc aac cat gag gat ggc ttctcc tca gcc ttc gtg ata gct 1563 Cys Ala Leu Asn His Glu Asp Gly Phe SerSer Ala Phe Val Ile Ala 385 390 395 cat gag acc ggc cac gtg ctc ggc atggag cat gac ggt cag ggg aat 1611 His Glu Thr Gly His Val Leu Gly Met GluHis Asp Gly Gln Gly Asn 400 405 410 ggc tgt gca gat gag acc agc ctg ggcagc gtc atg gcg ccc ctg gtg 1659 Gly Cys Ala Asp Glu Thr Ser Leu Gly SerVal Met Ala Pro Leu Val 415 420 425 cag gct gcc ttc cac cgc ttc cat tggtcc cgc tgc agc aag ctg gag 1707 Gln Ala Ala Phe His Arg Phe His Trp SerArg Cys Ser Lys Leu Glu 430 435 440 445 ctc agc cgc tac ctc ccc tcc tacgac tgc ctc ctc gat gac ccc ttt 1755 Leu Ser Arg Tyr Leu Pro Ser Tyr AspCys Leu Leu Asp Asp Pro Phe 450 455 460 gat cct gcc tgg ccc cag ccc ccagag ctg cct ggg atc aac tac tca 1803 Asp Pro Ala Trp Pro Gln Pro Pro GluLeu Pro Gly Ile Asn Tyr Ser 465 470 475 atg gat gag cag tgc cgc ttt gacttt ggc agt ggc tac cag acc tgc 1851 Met Asp Glu Gln Cys Arg Phe Asp PheGly Ser Gly Tyr Gln Thr Cys 480 485 490 ttg gca atc agg acc ttt gag ccctgc aag cag ctg tgg tgc agc cat 1899 Leu Ala Ile Arg Thr Phe Glu Pro CysLys Gln Leu Trp Cys Ser His 495 500 505 cct gac aac ccg tac ttc tgc aagacc aag aag ggg ccc ccg ctg gat 1947 Pro Asp Asn Pro Tyr Phe Cys Lys ThrLys Lys Gly Pro Pro Leu Asp 510 515 520 525 ggg act gag tgt gca ccc ggcaag tgg tgc ttc aaa ggt cac tgc atc 1995 Gly Thr Glu Cys Ala Pro Gly LysTrp Cys Phe Lys Gly His Cys Ile 530 535 540 tgg aag tcg ccg gag cag acatat ggc cag gat gga ggc tgg agc tcc 2043 Trp Lys Ser Pro Glu Gln Thr TyrGly Gln Asp Gly Gly Trp Ser Ser 545 550 555 tgg acc aag ttt ggg tca tgttcg cgg tca tgt ggg ggc ggg gtg cga 2091 Trp Thr Lys Phe Gly Ser Cys SerArg Ser Cys Gly Gly Gly Val Arg 560 565 570 tcc cgc agc cgg agc tgc aacaac ccc tcc cca gcc tat gga ggc cgc 2139 Ser Arg Ser Arg Ser Cys Asn AsnPro Ser Pro Ala Tyr Gly Gly Arg 575 580 585 ccg tgc tta ggg ccc atg ttcgag tac cag gtc tgc aac agc gag gag 2187 Pro Cys Leu Gly Pro Met Phe GluTyr Gln Val Cys Asn Ser Glu Glu 590 595 600 605 tgc cct ggg acc tac gaggac ttc cgg gcc cag cag tgt gcc aag cgc 2235 Cys Pro Gly Thr Tyr Glu AspPhe Arg Ala Gln Gln Cys Ala Lys Arg 610 615 620 aac tcg tac tat gtg caccag aat gcc aag cac agc tgg gtg ccc tac 2283 Asn Ser Tyr Tyr Val His GlnAsn Ala Lys His Ser Trp Val Pro Tyr 625 630 635 gag cct gac gat gac gcccag aag tgt gag ctg atc tgc cag tcg gcg 2331 Glu Pro Asp Asp Asp Ala GlnLys Cys Glu Leu Ile Cys Gln Ser Ala 640 645 650 gac aca ggg gac gtg gtgttc atg aac cag gtg gtt cac gat ggg aca 2379 Asp Thr Gly Asp Val Val PheMet Asn Gln Val Val His Asp Gly Thr 655 660 665 cgc tgc agc tac cgg gaccca tac agc gtc tgt gcg cgt ggc gag tgt 2427 Arg Cys Ser Tyr Arg Asp ProTyr Ser Val Cys Ala Arg Gly Glu Cys 670 675 680 685 gtg cct gtc ggc tgtgac aag gag gtg ggg tcc atg aag gcg gat gac 2475 Val Pro Val Gly Cys AspLys Glu Val Gly Ser Met Lys Ala Asp Asp 690 695 700 aag tgt gga gtc tgcggg ggt gac aac tcc cac tgc agg act gtg aag 2523 Lys Cys Gly Val Cys GlyGly Asp Asn Ser His Cys Arg Thr Val Lys 705 710 715 ggg acg ctg ggc aaggcc tcc aag cag gca gga gct ctc aag ctg gtg 2571 Gly Thr Leu Gly Lys AlaSer Lys Gln Ala Gly Ala Leu Lys Leu Val 720 725 730 cag atc cca gca ggtgcc agg cac atc cag att gag gca ctg gag aag 2619 Gln Ile Pro Ala Gly AlaArg His Ile Gln Ile Glu Ala Leu Glu Lys 735 740 745 tcc ccc cac cgc attgtg gtg aag aac cag gtc acc ggc agc ttc atc 2667 Ser Pro His Arg Ile ValVal Lys Asn Gln Val Thr Gly Ser Phe Ile 750 755 760 765 ctc aac ccc aagggc aag gaa gcc aca agc cgg acc ttc acc gcc atg 2715 Leu Asn Pro Lys GlyLys Glu Ala Thr Ser Arg Thr Phe Thr Ala Met 770 775 780 ggc ctg gag tgggag gat gcg gtg gag gat gcc aag gaa agc ttc aag 2763 Gly Leu Glu Trp GluAsp Ala Val Glu Asp Ala Lys Glu Ser Phe Lys 785 790 795 acc agc ggg cccctg cct gaa gcc att gcc atc ctg gct ctc ccc cca 2811 Thr Ser Gly Pro LeuPro Glu Ala Ile Ala Ile Leu Ala Leu Pro Pro 800 805 810 act gag ggt ggcccc cgc agc agc ctg gcc tac aag tac gtc atc cat 2859 Thr Glu Gly Gly ProArg Ser Ser Leu Ala Tyr Lys Tyr Val Ile His 815 820 825 gag gac ctg ctgccc ctt atc ggg agc aac aat gtg ctc ctg gag gag 2907 Glu Asp Leu Leu ProLeu Ile Gly Ser Asn Asn Val Leu Leu Glu Glu 830 835 840 845 atg gac acctat gag tgg gcg ctc aag agc tgg gcc ccc tgc agc aag 2955 Met Asp Thr TyrGlu Trp Ala Leu Lys Ser Trp Ala Pro Cys Ser Lys 850 855 860 gcc tgt ggagga ggg atc cag ttc acc aaa tac ggc tgc cgg cgc aga 3003 Ala Cys Gly GlyGly Ile Gln Phe Thr Lys Tyr Gly Cys Arg Arg Arg 865 870 875 cga gac caccac atg gtg cag cga cac ctg tgt gac cac aag aag agg 3051 Arg Asp His HisMet Val Gln Arg His Leu Cys Asp His Lys Lys Arg 880 885 890 ccc aag cccatc cgc cgg cgc tgc aac cag cac ccg tgc tct cag cct 3099 Pro Lys Pro IleArg Arg Arg Cys Asn Gln His Pro Cys Ser Gln Pro 895 900 905 gtg tgg gtgacg gag gag tgg ggt gcc tgc agc cgg agc tgt ggg aag 3147 Val Trp Val ThrGlu Glu Trp Gly Ala Cys Ser Arg Ser Cys Gly Lys 910 915 920 925 ctg ggggtg cag aca cgg ggg ata cag tgc ctg atg ccc ctc tcc aat 3195 Leu Gly ValGln Thr Arg Gly Ile Gln Cys Leu Met Pro Leu Ser Asn 930 935 940 gga acccac aag gtc atg ccg gcc aaa gcc tgt gcc ggg gac cgg cct 3243 Gly Thr HisLys Val Met Pro Ala Lys Ala Cys Ala Gly Asp Arg Pro 945 950 955 gag gcccga cgg ccc tgt ctc cga gtg ccc tgc cca gcc cag tgg agg 3291 Glu Ala ArgArg Pro Cys Leu Arg Val Pro Cys Pro Ala Gln Trp Arg 960 965 970 ctg ggagcc tgg tcc cag tgc tct gcc acc tgt gga gag ggc atc cag 3339 Leu Gly AlaTrp Ser Gln Cys Ser Ala Thr Cys Gly Glu Gly Ile Gln 975 980 985 cag cggcag gtg gtg tgc agg acc aac gcc aac agc ctc ggg cat tgc 3387 Gln Arg GlnVal Val Cys Arg Thr Asn Ala Asn Ser Leu Gly His Cys 990 995 1000 1005gag ggg gat agg cca gac act gtc cag gtc tgc agc ctg ccc gcc tgt 3435 GluGly Asp Arg Pro Asp Thr Val Gln Val Cys Ser Leu Pro Ala Cys 1010 10151020 gga gga aat cac cag aac tcc acg gtg agg gcc gat gtc tgg gaa ctt3483 Gly Gly Asn His Gln Asn Ser Thr Val Arg Ala Asp Val Trp Glu Leu1025 1030 1035 ggg acg cca gag ggg cag tgg gtg cca caa tct gaa ccc ctacat ccc 3531 Gly Thr Pro Glu Gly Gln Trp Val Pro Gln Ser Glu Pro Leu HisPro 1040 1045 1050 att aac aag ata tca tca acg gag ccc tgc acg gga gacagg tct gtc 3579 Ile Asn Lys Ile Ser Ser Thr Glu Pro Cys Thr Gly Asp ArgSer Val 1055 1060 1065 ttc tgc cag atg gaa gtg ctc gat cgc tac tgc tccatt ccc ggc tac 3627 Phe Cys Gln Met Glu Val Leu Asp Arg Tyr Cys Ser IlePro Gly Tyr 1070 1075 1080 1085 cac cgg ctc tgc tgt gtg tcc tgc atc aagaag gcc tcg ggc ccc aac 3675 His Arg Leu Cys Cys Val Ser Cys Ile Lys LysAla Ser Gly Pro Asn 1090 1095 1100 cct ggc cca gac cct ggc cca acc tcactg ccc ccc ttc tcc act cct 3723 Pro Gly Pro Asp Pro Gly Pro Thr Ser LeuPro Pro Phe Ser Thr Pro 1105 1110 1115 gga agc ccc tta cca gga ccc caggac cct gca gat gct gca gag cct 3771 Gly Ser Pro Leu Pro Gly Pro Gln AspPro Ala Asp Ala Ala Glu Pro 1120 1125 1130 cct gga aag cca acg gga tcagag gac cat cag cat ggc cga gcc aca 3819 Pro Gly Lys Pro Thr Gly Ser GluAsp His Gln His Gly Arg Ala Thr 1135 1140 1145 cag ctc cca gga gct ctggat aca agc tcc cca ggg acc cag cat ccc 3867 Gln Leu Pro Gly Ala Leu AspThr Ser Ser Pro Gly Thr Gln His Pro 1150 1155 1160 1165 ttt gcc cct gagaca cca atc cct gga gca tcc tgg agc atc tcc cct 3915 Phe Ala Pro Glu ThrPro Ile Pro Gly Ala Ser Trp Ser Ile Ser Pro 1170 1175 1180 acc acc cccggg ggg ctg cct tgg ggc tgg act cag aca cct acg cca 3963 Thr Thr Pro GlyGly Leu Pro Trp Gly Trp Thr Gln Thr Pro Thr Pro 1185 1190 1195 gtc cctgag gac aaa ggg caa cct gga gaa gac ctg aga cat ccc ggc 4011 Val Pro GluAsp Lys Gly Gln Pro Gly Glu Asp Leu Arg His Pro Gly 1200 1205 1210 accagc ctc cct gct gcc tcc ccg gtg aca tga gctgtgccct gccatcccac 4064 ThrSer Leu Pro Ala Ala Ser Pro Val Thr * 1215 1220 tggcacgttt acactctgtgtactgccccg tgactcccag ctcagaggac acacatagca 4124 gggcaggcgc aagcacagacttcattttaa atcattcgcc ttcttctcgt ttggggctgt 4184 gatgctcttt accccacaaagcggggtggg aggaagacaa agatcaggga aagccctaat 4244 cggagatacc tcagcaagctgcccccggcg ggactgaccc tctcagggcc cctgttggtc 4304 tcccctgcca agaccagggtcaactattgc tccctcctca cagaccctgg gcctgggcag 4364 atctgaatcc cggctggtctgtagctagaa gctgtcaggg ctgcctgcct tcccggaact 4424 gtgaggaccc ctgtggaggccctgcatatt tggcccctct ccccagaaag gcaaagcagg 4484 gccagggtag gtgggggactgttcacagcc aggccgagag gaggggggcc tgggaatgtg 4544 gcatgaggct tcccagctgcagggctggag ggggtggaac acaagatgat cgcaggccca 4604 gctcccggaa gccaagagctccatgcagtt ccaccagctg aggccaggca gcagaggcca 4664 gtttgtcttt gctggccagaagatggtgct catggccata ctctggcctt gcagatgtca 4724 ctagtgttac ttctagtgagtccagattac agactggccc cccaatctca ccccagccca 4784 ccagagaagg gggctcaggacaccctggac cccaagtcct cagcatccag ggatttccaa 4844 actggcgctc accccctgactccaccagga tggcaacttc aattatcact ctcagcctgg 4904 aaggggactc tgtgggacacagagggaaca cgatttctca ggctgtccct tcaatcattg 4964 cccttctccg aagatcgctcctgctggagt cggacatctt catcttctac ctggctcaag 5024 ctgggccaga gtgtgtggttctcccagggg tggttggacc ccaggactga ggaccagagt 5084 ctactcatag cctggccctggagatgacaa gggccaccca ggccaagtgc cccagggcag 5144 ggtgccagcc cctggcctggtgctggagtg gggaagacac actcacccac ggtgctgtaa 5204 gggcctgagc tgtgctcagctgccggccat gctacctcca agggacaggt aacagtctta 5264 gatcctctgg ctctcaggaagtggcagggg gtcccaggac acctccgggg tcttggagga 5324 tgtctcctaa actcctgccaggtgatagag gtgcttctca cttcttcctt ccccaaggca 5384 aaggggctgt tctgagccagcctggaggaa catgagtagt gggcccctgg cctgcaaccc 5444 ctttggagag tggaggtcctggggggctcc ccgccctccc cctgttgccc tcccctccct 5504 gggatgctgg ggcacacgtggagtcattcc tgtgagaacc agcctggcct gtgttaaact 5564 cttgtgcctt ggaaatccagatctttaaaa ttttatgtat ttattaacat cgccattggg 5624 ccccaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5684 aaaaaagggg cggccgc 57012 1223 PRT Homo sapiens 2 Met Ala Pro Leu Arg Ala Leu Leu Ser Tyr LeuLeu Pro Leu His Cys 1 5 10 15 Ala Leu Cys Ala Ala Ala Gly Ser Arg ThrPro Glu Leu His Leu Ser 20 25 30 Gly Lys Leu Ser Asp Tyr Gly Val Thr ValPro Cys Ser Thr Asp Phe 35 40 45 Arg Gly Arg Phe Leu Ser His Val Val SerGly Pro Ala Ala Ala Ser 50 55 60 Ala Gly Ser Met Val Val Asp Thr Pro ProThr Leu Pro Arg His Ser 65 70 75 80 Ser His Leu Arg Val Ala Arg Ser ProLeu His Pro Gly Gly Thr Leu 85 90 95 Trp Pro Gly Arg Val Gly Arg His SerLeu Tyr Phe Asn Val Thr Val 100 105 110 Phe Gly Lys Glu Leu His Leu ArgLeu Arg Pro Asn Arg Arg Leu Val 115 120 125 Val Pro Gly Ser Ser Val GluTrp Gln Glu Asp Phe Arg Glu Leu Phe 130 135 140 Arg Gln Pro Leu Arg GlnGlu Cys Val Tyr Thr Gly Gly Val Thr Gly 145 150 155 160 Met Pro Gly AlaAla Val Ala Ile Ser Asn Cys Asp Gly Leu Ala Gly 165 170 175 Leu Ile ArgThr Asp Ser Thr Asp Phe Phe Ile Glu Pro Leu Glu Arg 180 185 190 Gly GlnGln Glu Lys Glu Ala Ser Gly Arg Thr His Val Val Tyr Arg 195 200 205 ArgGlu Ala Val Gln Gln Glu Trp Ala Glu Pro Asp Gly Asp Leu His 210 215 220Asn Glu Ala Phe Gly Leu Gly Asp Leu Pro Asn Leu Leu Gly Leu Val 225 230235 240 Gly Asp Gln Leu Gly Asp Thr Glu Arg Lys Arg Arg His Ala Lys Pro245 250 255 Gly Ser Tyr Ser Ile Glu Val Leu Leu Val Val Asp Asp Ser ValVal 260 265 270 Arg Phe His Gly Lys Glu His Val Gln Asn Tyr Val Leu ThrLeu Met 275 280 285 Asn Ile Val Asp Glu Ile Tyr His Asp Glu Ser Leu GlyVal His Ile 290 295 300 Asn Ile Ala Leu Val Arg Leu Ile Met Val Gly TyrArg Gln Ser Leu 305 310 315 320 Ser Leu Ile Glu Arg Gly Asn Pro Ser ArgSer Leu Glu Gln Val Cys 325 330 335 Arg Trp Ala His Ser Gln Gln Arg GlnAsp Pro Ser His Ala Glu His 340 345 350 His Asp His Val Val Phe Leu ThrArg Gln Asp Phe Gly Pro Ser Gly 355 360 365 Tyr Ala Pro Val Thr Gly MetCys His Pro Leu Arg Ser Cys Ala Leu 370 375 380 Asn His Glu Asp Gly PheSer Ser Ala Phe Val Ile Ala His Glu Thr 385 390 395 400 Gly His Val LeuGly Met Glu His Asp Gly Gln Gly Asn Gly Cys Ala 405 410 415 Asp Glu ThrSer Leu Gly Ser Val Met Ala Pro Leu Val Gln Ala Ala 420 425 430 Phe HisArg Phe His Trp Ser Arg Cys Ser Lys Leu Glu Leu Ser Arg 435 440 445 TyrLeu Pro Ser Tyr Asp Cys Leu Leu Asp Asp Pro Phe Asp Pro Ala 450 455 460Trp Pro Gln Pro Pro Glu Leu Pro Gly Ile Asn Tyr Ser Met Asp Glu 465 470475 480 Gln Cys Arg Phe Asp Phe Gly Ser Gly Tyr Gln Thr Cys Leu Ala Ile485 490 495 Arg Thr Phe Glu Pro Cys Lys Gln Leu Trp Cys Ser His Pro AspAsn 500 505 510 Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro Pro Leu Asp GlyThr Glu 515 520 525 Cys Ala Pro Gly Lys Trp Cys Phe Lys Gly His Cys IleTrp Lys Ser 530 535 540 Pro Glu Gln Thr Tyr Gly Gln Asp Gly Gly Trp SerSer Trp Thr Lys 545 550 555 560 Phe Gly Ser Cys Ser Arg Ser Cys Gly GlyGly Val Arg Ser Arg Ser 565 570 575 Arg Ser Cys Asn Asn Pro Ser Pro AlaTyr Gly Gly Arg Pro Cys Leu 580 585 590 Gly Pro Met Phe Glu Tyr Gln ValCys Asn Ser Glu Glu Cys Pro Gly 595 600 605 Thr Tyr Glu Asp Phe Arg AlaGln Gln Cys Ala Lys Arg Asn Ser Tyr 610 615 620 Tyr Val His Gln Asn AlaLys His Ser Trp Val Pro Tyr Glu Pro Asp 625 630 635 640 Asp Asp Ala GlnLys Cys Glu Leu Ile Cys Gln Ser Ala Asp Thr Gly 645 650 655 Asp Val ValPhe Met Asn Gln Val Val His Asp Gly Thr Arg Cys Ser 660 665 670 Tyr ArgAsp Pro Tyr Ser Val Cys Ala Arg Gly Glu Cys Val Pro Val 675 680 685 GlyCys Asp Lys Glu Val Gly Ser Met Lys Ala Asp Asp Lys Cys Gly 690 695 700Val Cys Gly Gly Asp Asn Ser His Cys Arg Thr Val Lys Gly Thr Leu 705 710715 720 Gly Lys Ala Ser Lys Gln Ala Gly Ala Leu Lys Leu Val Gln Ile Pro725 730 735 Ala Gly Ala Arg His Ile Gln Ile Glu Ala Leu Glu Lys Ser ProHis 740 745 750 Arg Ile Val Val Lys Asn Gln Val Thr Gly Ser Phe Ile LeuAsn Pro 755 760 765 Lys Gly Lys Glu Ala Thr Ser Arg Thr Phe Thr Ala MetGly Leu Glu 770 775 780 Trp Glu Asp Ala Val Glu Asp Ala Lys Glu Ser PheLys Thr Ser Gly 785 790 795 800 Pro Leu Pro Glu Ala Ile Ala Ile Leu AlaLeu Pro Pro Thr Glu Gly 805 810 815 Gly Pro Arg Ser Ser Leu Ala Tyr LysTyr Val Ile His Glu Asp Leu 820 825 830 Leu Pro Leu Ile Gly Ser Asn AsnVal Leu Leu Glu Glu Met Asp Thr 835 840 845 Tyr Glu Trp Ala Leu Lys SerTrp Ala Pro Cys Ser Lys Ala Cys Gly 850 855 860 Gly Gly Ile Gln Phe ThrLys Tyr Gly Cys Arg Arg Arg Arg Asp His 865 870 875 880 His Met Val GlnArg His Leu Cys Asp His Lys Lys Arg Pro Lys Pro 885 890 895 Ile Arg ArgArg Cys Asn Gln His Pro Cys Ser Gln Pro Val Trp Val 900 905 910 Thr GluGlu Trp Gly Ala Cys Ser Arg Ser Cys Gly Lys Leu Gly Val 915 920 925 GlnThr Arg Gly Ile Gln Cys Leu Met Pro Leu Ser Asn Gly Thr His 930 935 940Lys Val Met Pro Ala Lys Ala Cys Ala Gly Asp Arg Pro Glu Ala Arg 945 950955 960 Arg Pro Cys Leu Arg Val Pro Cys Pro Ala Gln Trp Arg Leu Gly Ala965 970 975 Trp Ser Gln Cys Ser Ala Thr Cys Gly Glu Gly Ile Gln Gln ArgGln 980 985 990 Val Val Cys Arg Thr Asn Ala Asn Ser Leu Gly His Cys GluGly Asp 995 1000 1005 Arg Pro Asp Thr Val Gln Val Cys Ser Leu Pro AlaCys Gly Gly Asn 1010 1015 1020 His Gln Asn Ser Thr Val Arg Ala Asp ValTrp Glu Leu Gly Thr Pro 1025 1030 1035 1040 Glu Gly Gln Trp Val Pro GlnSer Glu Pro Leu His Pro Ile Asn Lys 1045 1050 1055 Ile Ser Ser Thr GluPro Cys Thr Gly Asp Arg Ser Val Phe Cys Gln 1060 1065 1070 Met Glu ValLeu Asp Arg Tyr Cys Ser Ile Pro Gly Tyr His Arg Leu 1075 1080 1085 CysCys Val Ser Cys Ile Lys Lys Ala Ser Gly Pro Asn Pro Gly Pro 1090 10951100 Asp Pro Gly Pro Thr Ser Leu Pro Pro Phe Ser Thr Pro Gly Ser Pro1105 1110 1115 1120 Leu Pro Gly Pro Gln Asp Pro Ala Asp Ala Ala Glu ProPro Gly Lys 1125 1130 1135 Pro Thr Gly Ser Glu Asp His Gln His Gly ArgAla Thr Gln Leu Pro 1140 1145 1150 Gly Ala Leu Asp Thr Ser Ser Pro GlyThr Gln His Pro Phe Ala Pro 1155 1160 1165 Glu Thr Pro Ile Pro Gly AlaSer Trp Ser Ile Ser Pro Thr Thr Pro 1170 1175 1180 Gly Gly Leu Pro TrpGly Trp Thr Gln Thr Pro Thr Pro Val Pro Glu 1185 1190 1195 1200 Asp LysGly Gln Pro Gly Glu Asp Leu Arg His Pro Gly Thr Ser Leu 1205 1210 1215Pro Ala Ala Ser Pro Val Thr 1220 3 3672 DNA Homo sapiens CDS(1)...(3672) 3 atg gct cca ctc cgc gcg ctg ctg tcc tac ctg ctg cct ttgcac tgt 48 Met Ala Pro Leu Arg Ala Leu Leu Ser Tyr Leu Leu Pro Leu HisCys 1 5 10 15 gcg ctc tgc gcc gcc gcg ggc agc cgg acc cca gag ctg cacctc tct 96 Ala Leu Cys Ala Ala Ala Gly Ser Arg Thr Pro Glu Leu His LeuSer 20 25 30 gga aag ctc agt gac tat ggt gtg aca gtg ccc tgc agc aca gacttt 144 Gly Lys Leu Ser Asp Tyr Gly Val Thr Val Pro Cys Ser Thr Asp Phe35 40 45 cgg gga cgc ttc ctc tcc cac gtg gtg tct ggc cca gca gca gcc tct192 Arg Gly Arg Phe Leu Ser His Val Val Ser Gly Pro Ala Ala Ala Ser 5055 60 gca ggg agc atg gta gtg gac acg cca ccc aca cta cca cga cac tcc240 Ala Gly Ser Met Val Val Asp Thr Pro Pro Thr Leu Pro Arg His Ser 6570 75 80 agt cac ctc cgg gtg gct cgc agc cct ctg cac cca gga ggg acc ctg288 Ser His Leu Arg Val Ala Arg Ser Pro Leu His Pro Gly Gly Thr Leu 8590 95 tgg cct ggc agg gtg ggg cgc cac tcc ctc tac ttc aat gtc act gtt336 Trp Pro Gly Arg Val Gly Arg His Ser Leu Tyr Phe Asn Val Thr Val 100105 110 ttc ggg aag gaa ctg cac ttg cgc ctg cgg ccc aat cgg agg ttg gta384 Phe Gly Lys Glu Leu His Leu Arg Leu Arg Pro Asn Arg Arg Leu Val 115120 125 gtg cca gga tcc tca gtg gag tgg cag gag gat ttt cgg gag ctg ttc432 Val Pro Gly Ser Ser Val Glu Trp Gln Glu Asp Phe Arg Glu Leu Phe 130135 140 cgg cag ccc tta cgg cag gag tgt gtg tac act gga ggt gtc act gga480 Arg Gln Pro Leu Arg Gln Glu Cys Val Tyr Thr Gly Gly Val Thr Gly 145150 155 160 atg cct ggg gca gct gtt gcc atc agc aac tgt gac gga ttg gcgggc 528 Met Pro Gly Ala Ala Val Ala Ile Ser Asn Cys Asp Gly Leu Ala Gly165 170 175 ctc atc cgc aca gac agc acc gac ttc ttc att gag cct ctg gagcgg 576 Leu Ile Arg Thr Asp Ser Thr Asp Phe Phe Ile Glu Pro Leu Glu Arg180 185 190 ggc cag cag gag aag gag gcc agc ggg agg aca cat gtg gtg taccgc 624 Gly Gln Gln Glu Lys Glu Ala Ser Gly Arg Thr His Val Val Tyr Arg195 200 205 cgg gag gcc gtc cag cag gag tgg gca gaa cct gac ggg gac ctgcac 672 Arg Glu Ala Val Gln Gln Glu Trp Ala Glu Pro Asp Gly Asp Leu His210 215 220 aat gaa gcc ttt ggc ctg gga gac ctt ccc aac ctg ctg ggc ctggtg 720 Asn Glu Ala Phe Gly Leu Gly Asp Leu Pro Asn Leu Leu Gly Leu Val225 230 235 240 ggg gac cag ctg ggc gac aca gag cgg aag cgg cgg cat gccaag cca 768 Gly Asp Gln Leu Gly Asp Thr Glu Arg Lys Arg Arg His Ala LysPro 245 250 255 ggc agc tac agc atc gag gtg ctg ctg gtg gtg gac gac tcggtg gtt 816 Gly Ser Tyr Ser Ile Glu Val Leu Leu Val Val Asp Asp Ser ValVal 260 265 270 cgc ttc cat ggc aag gag cat gtg cag aac tat gtc ctc accctc atg 864 Arg Phe His Gly Lys Glu His Val Gln Asn Tyr Val Leu Thr LeuMet 275 280 285 aat atc gta gat gag att tac cac gat gag tcc ctg ggg gttcat ata 912 Asn Ile Val Asp Glu Ile Tyr His Asp Glu Ser Leu Gly Val HisIle 290 295 300 aat att gcc ctc gtc cgc ttg atc atg gtt ggc tac cga cagtcc ctg 960 Asn Ile Ala Leu Val Arg Leu Ile Met Val Gly Tyr Arg Gln SerLeu 305 310 315 320 agc ctg atc gag cgc ggg aac ccc tca cgc agc ctg gagcag gtg tgt 1008 Ser Leu Ile Glu Arg Gly Asn Pro Ser Arg Ser Leu Glu GlnVal Cys 325 330 335 cgc tgg gca cac tcc cag cag cgc cag gac ccc agc cacgct gag cac 1056 Arg Trp Ala His Ser Gln Gln Arg Gln Asp Pro Ser His AlaGlu His 340 345 350 cat gac cac gtt gtg ttc ctc acc cgg cag gac ttt gggccc tca ggg 1104 His Asp His Val Val Phe Leu Thr Arg Gln Asp Phe Gly ProSer Gly 355 360 365 tat gca ccc gtc act ggc atg tgt cac ccc ctg agg agctgt gcc ctc 1152 Tyr Ala Pro Val Thr Gly Met Cys His Pro Leu Arg Ser CysAla Leu 370 375 380 aac cat gag gat ggc ttc tcc tca gcc ttc gtg ata gctcat gag acc 1200 Asn His Glu Asp Gly Phe Ser Ser Ala Phe Val Ile Ala HisGlu Thr 385 390 395 400 ggc cac gtg ctc ggc atg gag cat gac ggt cag gggaat ggc tgt gca 1248 Gly His Val Leu Gly Met Glu His Asp Gly Gln Gly AsnGly Cys Ala 405 410 415 gat gag acc agc ctg ggc agc gtc atg gcg ccc ctggtg cag gct gcc 1296 Asp Glu Thr Ser Leu Gly Ser Val Met Ala Pro Leu ValGln Ala Ala 420 425 430 ttc cac cgc ttc cat tgg tcc cgc tgc agc aag ctggag ctc agc cgc 1344 Phe His Arg Phe His Trp Ser Arg Cys Ser Lys Leu GluLeu Ser Arg 435 440 445 tac ctc ccc tcc tac gac tgc ctc ctc gat gac cccttt gat cct gcc 1392 Tyr Leu Pro Ser Tyr Asp Cys Leu Leu Asp Asp Pro PheAsp Pro Ala 450 455 460 tgg ccc cag ccc cca gag ctg cct ggg atc aac tactca atg gat gag 1440 Trp Pro Gln Pro Pro Glu Leu Pro Gly Ile Asn Tyr SerMet Asp Glu 465 470 475 480 cag tgc cgc ttt gac ttt ggc agt ggc tac cagacc tgc ttg gca atc 1488 Gln Cys Arg Phe Asp Phe Gly Ser Gly Tyr Gln ThrCys Leu Ala Ile 485 490 495 agg acc ttt gag ccc tgc aag cag ctg tgg tgcagc cat cct gac aac 1536 Arg Thr Phe Glu Pro Cys Lys Gln Leu Trp Cys SerHis Pro Asp Asn 500 505 510 ccg tac ttc tgc aag acc aag aag ggg ccc ccgctg gat ggg act gag 1584 Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro Pro LeuAsp Gly Thr Glu 515 520 525 tgt gca ccc ggc aag tgg tgc ttc aaa ggt cactgc atc tgg aag tcg 1632 Cys Ala Pro Gly Lys Trp Cys Phe Lys Gly His CysIle Trp Lys Ser 530 535 540 ccg gag cag aca tat ggc cag gat gga ggc tggagc tcc tgg acc aag 1680 Pro Glu Gln Thr Tyr Gly Gln Asp Gly Gly Trp SerSer Trp Thr Lys 545 550 555 560 ttt ggg tca tgt tcg cgg tca tgt ggg ggcggg gtg cga tcc cgc agc 1728 Phe Gly Ser Cys Ser Arg Ser Cys Gly Gly GlyVal Arg Ser Arg Ser 565 570 575 cgg agc tgc aac aac ccc tcc cca gcc tatgga ggc cgc ccg tgc tta 1776 Arg Ser Cys Asn Asn Pro Ser Pro Ala Tyr GlyGly Arg Pro Cys Leu 580 585 590 ggg ccc atg ttc gag tac cag gtc tgc aacagc gag gag tgc cct ggg 1824 Gly Pro Met Phe Glu Tyr Gln Val Cys Asn SerGlu Glu Cys Pro Gly 595 600 605 acc tac gag gac ttc cgg gcc cag cag tgtgcc aag cgc aac tcg tac 1872 Thr Tyr Glu Asp Phe Arg Ala Gln Gln Cys AlaLys Arg Asn Ser Tyr 610 615 620 tat gtg cac cag aat gcc aag cac agc tgggtg ccc tac gag cct gac 1920 Tyr Val His Gln Asn Ala Lys His Ser Trp ValPro Tyr Glu Pro Asp 625 630 635 640 gat gac gcc cag aag tgt gag ctg atctgc cag tcg gcg gac aca ggg 1968 Asp Asp Ala Gln Lys Cys Glu Leu Ile CysGln Ser Ala Asp Thr Gly 645 650 655 gac gtg gtg ttc atg aac cag gtg gttcac gat ggg aca cgc tgc agc 2016 Asp Val Val Phe Met Asn Gln Val Val HisAsp Gly Thr Arg Cys Ser 660 665 670 tac cgg gac cca tac agc gtc tgt gcgcgt ggc gag tgt gtg cct gtc 2064 Tyr Arg Asp Pro Tyr Ser Val Cys Ala ArgGly Glu Cys Val Pro Val 675 680 685 ggc tgt gac aag gag gtg ggg tcc atgaag gcg gat gac aag tgt gga 2112 Gly Cys Asp Lys Glu Val Gly Ser Met LysAla Asp Asp Lys Cys Gly 690 695 700 gtc tgc ggg ggt gac aac tcc cac tgcagg act gtg aag ggg acg ctg 2160 Val Cys Gly Gly Asp Asn Ser His Cys ArgThr Val Lys Gly Thr Leu 705 710 715 720 ggc aag gcc tcc aag cag gca ggagct ctc aag ctg gtg cag atc cca 2208 Gly Lys Ala Ser Lys Gln Ala Gly AlaLeu Lys Leu Val Gln Ile Pro 725 730 735 gca ggt gcc agg cac atc cag attgag gca ctg gag aag tcc ccc cac 2256 Ala Gly Ala Arg His Ile Gln Ile GluAla Leu Glu Lys Ser Pro His 740 745 750 cgc att gtg gtg aag aac cag gtcacc ggc agc ttc atc ctc aac ccc 2304 Arg Ile Val Val Lys Asn Gln Val ThrGly Ser Phe Ile Leu Asn Pro 755 760 765 aag ggc aag gaa gcc aca agc cggacc ttc acc gcc atg ggc ctg gag 2352 Lys Gly Lys Glu Ala Thr Ser Arg ThrPhe Thr Ala Met Gly Leu Glu 770 775 780 tgg gag gat gcg gtg gag gat gccaag gaa agc ttc aag acc agc ggg 2400 Trp Glu Asp Ala Val Glu Asp Ala LysGlu Ser Phe Lys Thr Ser Gly 785 790 795 800 ccc ctg cct gaa gcc att gccatc ctg gct ctc ccc cca act gag ggt 2448 Pro Leu Pro Glu Ala Ile Ala IleLeu Ala Leu Pro Pro Thr Glu Gly 805 810 815 ggc ccc cgc agc agc ctg gcctac aag tac gtc atc cat gag gac ctg 2496 Gly Pro Arg Ser Ser Leu Ala TyrLys Tyr Val Ile His Glu Asp Leu 820 825 830 ctg ccc ctt atc ggg agc aacaat gtg ctc ctg gag gag atg gac acc 2544 Leu Pro Leu Ile Gly Ser Asn AsnVal Leu Leu Glu Glu Met Asp Thr 835 840 845 tat gag tgg gcg ctc aag agctgg gcc ccc tgc agc aag gcc tgt gga 2592 Tyr Glu Trp Ala Leu Lys Ser TrpAla Pro Cys Ser Lys Ala Cys Gly 850 855 860 gga ggg atc cag ttc acc aaatac ggc tgc cgg cgc aga cga gac cac 2640 Gly Gly Ile Gln Phe Thr Lys TyrGly Cys Arg Arg Arg Arg Asp His 865 870 875 880 cac atg gtg cag cga cacctg tgt gac cac aag aag agg ccc aag ccc 2688 His Met Val Gln Arg His LeuCys Asp His Lys Lys Arg Pro Lys Pro 885 890 895 atc cgc cgg cgc tgc aaccag cac ccg tgc tct cag cct gtg tgg gtg 2736 Ile Arg Arg Arg Cys Asn GlnHis Pro Cys Ser Gln Pro Val Trp Val 900 905 910 acg gag gag tgg ggt gcctgc agc cgg agc tgt ggg aag ctg ggg gtg 2784 Thr Glu Glu Trp Gly Ala CysSer Arg Ser Cys Gly Lys Leu Gly Val 915 920 925 cag aca cgg ggg ata cagtgc ctg atg ccc ctc tcc aat gga acc cac 2832 Gln Thr Arg Gly Ile Gln CysLeu Met Pro Leu Ser Asn Gly Thr His 930 935 940 aag gtc atg ccg gcc aaagcc tgt gcc ggg gac cgg cct gag gcc cga 2880 Lys Val Met Pro Ala Lys AlaCys Ala Gly Asp Arg Pro Glu Ala Arg 945 950 955 960 cgg ccc tgt ctc cgagtg ccc tgc cca gcc cag tgg agg ctg gga gcc 2928 Arg Pro Cys Leu Arg ValPro Cys Pro Ala Gln Trp Arg Leu Gly Ala 965 970 975 tgg tcc cag tgc tctgcc acc tgt gga gag ggc atc cag cag cgg cag 2976 Trp Ser Gln Cys Ser AlaThr Cys Gly Glu Gly Ile Gln Gln Arg Gln 980 985 990 gtg gtg tgc agg accaac gcc aac agc ctc ggg cat tgc gag ggg gat 3024 Val Val Cys Arg Thr AsnAla Asn Ser Leu Gly His Cys Glu Gly Asp 995 1000 1005 agg cca gac actgtc cag gtc tgc agc ctg ccc gcc tgt gga gga aat 3072 Arg Pro Asp Thr ValGln Val Cys Ser Leu Pro Ala Cys Gly Gly Asn 1010 1015 1020 cac cag aactcc acg gtg agg gcc gat gtc tgg gaa ctt ggg acg cca 3120 His Gln Asn SerThr Val Arg Ala Asp Val Trp Glu Leu Gly Thr Pro 1025 1030 1035 1040 gagggg cag tgg gtg cca caa tct gaa ccc cta cat ccc att aac aag 3168 Glu GlyGln Trp Val Pro Gln Ser Glu Pro Leu His Pro Ile Asn Lys 1045 1050 1055ata tca tca acg gag ccc tgc acg gga gac agg tct gtc ttc tgc cag 3216 IleSer Ser Thr Glu Pro Cys Thr Gly Asp Arg Ser Val Phe Cys Gln 1060 10651070 atg gaa gtg ctc gat cgc tac tgc tcc att ccc ggc tac cac cgg ctc3264 Met Glu Val Leu Asp Arg Tyr Cys Ser Ile Pro Gly Tyr His Arg Leu1075 1080 1085 tgc tgt gtg tcc tgc atc aag aag gcc tcg ggc ccc aac cctggc cca 3312 Cys Cys Val Ser Cys Ile Lys Lys Ala Ser Gly Pro Asn Pro GlyPro 1090 1095 1100 gac cct ggc cca acc tca ctg ccc ccc ttc tcc act cctgga agc ccc 3360 Asp Pro Gly Pro Thr Ser Leu Pro Pro Phe Ser Thr Pro GlySer Pro 1105 1110 1115 1120 tta cca gga ccc cag gac cct gca gat gct gcagag cct cct gga aag 3408 Leu Pro Gly Pro Gln Asp Pro Ala Asp Ala Ala GluPro Pro Gly Lys 1125 1130 1135 cca acg gga tca gag gac cat cag cat ggccga gcc aca cag ctc cca 3456 Pro Thr Gly Ser Glu Asp His Gln His Gly ArgAla Thr Gln Leu Pro 1140 1145 1150 gga gct ctg gat aca agc tcc cca gggacc cag cat ccc ttt gcc cct 3504 Gly Ala Leu Asp Thr Ser Ser Pro Gly ThrGln His Pro Phe Ala Pro 1155 1160 1165 gag aca cca atc cct gga gca tcctgg agc atc tcc cct acc acc ccc 3552 Glu Thr Pro Ile Pro Gly Ala Ser TrpSer Ile Ser Pro Thr Thr Pro 1170 1175 1180 ggg ggg ctg cct tgg ggc tggact cag aca cct acg cca gtc cct gag 3600 Gly Gly Leu Pro Trp Gly Trp ThrGln Thr Pro Thr Pro Val Pro Glu 1185 1190 1195 1200 gac aaa ggg caa cctgga gaa gac ctg aga cat ccc ggc acc agc ctc 3648 Asp Lys Gly Gln Pro GlyGlu Asp Leu Arg His Pro Gly Thr Ser Leu 1205 1210 1215 cct gct gcc tccccg gtg aca tga 3672 Pro Ala Ala Ser Pro Val Thr * 1220 4 5344 DNA Homosapiens CDS (218)...(3688) 4 tcccccgggc tgcaggaatt cggcacgagg cgcgctgctgtcctacctgc tgcctttgca 60 ctgtgcgctc tgcgccgccg cgggcagccg gaccccagagctgcacctct ctggaaagct 120 cagtgactat ggtgtgacag tgccctgcag cacagactttcggggacgct tcctctccca 180 cgtggtgtct ggcccagcag cagcctctgc agggagc atggta gtg gac acg cca 235 Met Val Val Asp Thr Pro 1 5 ccc aca cta cca cgacac tcc agt cac ctc cgg gtg gct cgc agc cct 283 Pro Thr Leu Pro Arg HisSer Ser His Leu Arg Val Ala Arg Ser Pro 10 15 20 ctg cac cca gga ggg accctg tgg cct ggc agg gtg ggg cgc cac tcc 331 Leu His Pro Gly Gly Thr LeuTrp Pro Gly Arg Val Gly Arg His Ser 25 30 35 ctc tac ttc aat gtc act gttttc ggg aag gaa ctg cac ttg cgc ctg 379 Leu Tyr Phe Asn Val Thr Val PheGly Lys Glu Leu His Leu Arg Leu 40 45 50 cgg ccc aat cgg agg ttg gta gtgcca gga tcc tca gtg gag tgg cag 427 Arg Pro Asn Arg Arg Leu Val Val ProGly Ser Ser Val Glu Trp Gln 55 60 65 70 gag gat ttt cgg gag ctg ttc cggcag ccc tta cgg cag gag tgt gtg 475 Glu Asp Phe Arg Glu Leu Phe Arg GlnPro Leu Arg Gln Glu Cys Val 75 80 85 tac act gga ggt gtc act gga atg cctggg gca gct gtt gcc atc agc 523 Tyr Thr Gly Gly Val Thr Gly Met Pro GlyAla Ala Val Ala Ile Ser 90 95 100 aac tgt gac gga ttg gcg ggc ctc atccgc aca gac agc acc gac ttc 571 Asn Cys Asp Gly Leu Ala Gly Leu Ile ArgThr Asp Ser Thr Asp Phe 105 110 115 ttc att gag cct ctg gag cgg ggc cagcag gag aag gag gcc agc ggg 619 Phe Ile Glu Pro Leu Glu Arg Gly Gln GlnGlu Lys Glu Ala Ser Gly 120 125 130 agg aca cat gtg gtg tac cgc cgg gaggcc gtc cag cag gag tgg gca 667 Arg Thr His Val Val Tyr Arg Arg Glu AlaVal Gln Gln Glu Trp Ala 135 140 145 150 gaa cct gac ggg gac ctg cac aatgaa gcc ttt ggc ctg gga gac ctt 715 Glu Pro Asp Gly Asp Leu His Asn GluAla Phe Gly Leu Gly Asp Leu 155 160 165 ccc aac ctg ctg ggc ctg gtg ggggac cag ctg ggc gac aca gag cgg 763 Pro Asn Leu Leu Gly Leu Val Gly AspGln Leu Gly Asp Thr Glu Arg 170 175 180 aag cgg cgg cat gcc aag cca ggcagc tac agc atc gag gtg ctg ctg 811 Lys Arg Arg His Ala Lys Pro Gly SerTyr Ser Ile Glu Val Leu Leu 185 190 195 gtg gtg gac gac tcg gtg gtt cgcttc cat ggc aag gag cat gtg cag 859 Val Val Asp Asp Ser Val Val Arg PheHis Gly Lys Glu His Val Gln 200 205 210 aac tat gtc ctc acc ctc atg aatatc gta gat gag att tac cac gat 907 Asn Tyr Val Leu Thr Leu Met Asn IleVal Asp Glu Ile Tyr His Asp 215 220 225 230 gag tcc ctg ggg gtt cat ataaat att gcc ctc gtc cgc ttg atc atg 955 Glu Ser Leu Gly Val His Ile AsnIle Ala Leu Val Arg Leu Ile Met 235 240 245 gtt ggc tac cga cag tcc ctgagc ctg atc gag cgc ggg aac ccc tca 1003 Val Gly Tyr Arg Gln Ser Leu SerLeu Ile Glu Arg Gly Asn Pro Ser 250 255 260 cgc agc ctg gag cag gtg tgtcgc tgg gca cac tcc cag cag cgc cag 1051 Arg Ser Leu Glu Gln Val Cys ArgTrp Ala His Ser Gln Gln Arg Gln 265 270 275 gac ccc agc cac gct gag caccat gac cac gtt gtg ttc ctc acc cgg 1099 Asp Pro Ser His Ala Glu His HisAsp His Val Val Phe Leu Thr Arg 280 285 290 cag gac ttt ggg ccc tca gggtat gca ccc gtc act ggc atg tgt cac 1147 Gln Asp Phe Gly Pro Ser Gly TyrAla Pro Val Thr Gly Met Cys His 295 300 305 310 ccc ctg agg agc tgt gccctc aac cat gag gat ggc ttc tcc tca gcc 1195 Pro Leu Arg Ser Cys Ala LeuAsn His Glu Asp Gly Phe Ser Ser Ala 315 320 325 ttc gtg ata gct cat gagacc ggc cac gtg ctc ggc atg gag cat gac 1243 Phe Val Ile Ala His Glu ThrGly His Val Leu Gly Met Glu His Asp 330 335 340 ggt cag ggg aat ggc tgtgca gat gag acc agc ctg ggc agc gtc atg 1291 Gly Gln Gly Asn Gly Cys AlaAsp Glu Thr Ser Leu Gly Ser Val Met 345 350 355 gcg ccc ctg gtg cag gctgcc ttc cac cgc ttc cat tgg tcc cgc tgc 1339 Ala Pro Leu Val Gln Ala AlaPhe His Arg Phe His Trp Ser Arg Cys 360 365 370 agc aag ctg gag ctc agccgc tac ctc ccc tcc tac gac tgc ctc ctc 1387 Ser Lys Leu Glu Leu Ser ArgTyr Leu Pro Ser Tyr Asp Cys Leu Leu 375 380 385 390 gat gac ccc ttt gatcct gcc tgg ccc cag ccc cca gag ctg cct ggg 1435 Asp Asp Pro Phe Asp ProAla Trp Pro Gln Pro Pro Glu Leu Pro Gly 395 400 405 atc aac tac tca atggat gag cag tgc cgc ttt gac ttt ggc agt ggc 1483 Ile Asn Tyr Ser Met AspGlu Gln Cys Arg Phe Asp Phe Gly Ser Gly 410 415 420 tac cag acc tgc ttggca atc agg acc ttt gag ccc tgc aag cag ctg 1531 Tyr Gln Thr Cys Leu AlaIle Arg Thr Phe Glu Pro Cys Lys Gln Leu 425 430 435 tgg tgc agc cat cctgac aac ccg tac ttc tgc aag acc aag aag ggg 1579 Trp Cys Ser His Pro AspAsn Pro Tyr Phe Cys Lys Thr Lys Lys Gly 440 445 450 ccc ccg ctg gat gggact gag tgt gca ccc ggc aag tgg tgc ttc aaa 1627 Pro Pro Leu Asp Gly ThrGlu Cys Ala Pro Gly Lys Trp Cys Phe Lys 455 460 465 470 ggt cac tgc atctgg aag tcg ccg gag cag aca tat ggc cag gat gga 1675 Gly His Cys Ile TrpLys Ser Pro Glu Gln Thr Tyr Gly Gln Asp Gly 475 480 485 ggc tgg agc tcctgg acc aag ttt ggg tca tgt tcg cgg tca tgt ggg 1723 Gly Trp Ser Ser TrpThr Lys Phe Gly Ser Cys Ser Arg Ser Cys Gly 490 495 500 ggc ggg gtg cgatcc cgc agc cgg agc tgc aac aac ccc tcc cca gcc 1771 Gly Gly Val Arg SerArg Ser Arg Ser Cys Asn Asn Pro Ser Pro Ala 505 510 515 tat gga ggc cgcccg tgc tta ggg ccc atg ttc gag tac cag gtc tgc 1819 Tyr Gly Gly Arg ProCys Leu Gly Pro Met Phe Glu Tyr Gln Val Cys 520 525 530 aac agc gag gagtgc cct ggg acc tac gag gac ttc cgg gcc cag cag 1867 Asn Ser Glu Glu CysPro Gly Thr Tyr Glu Asp Phe Arg Ala Gln Gln 535 540 545 550 tgt gcc aagcgc aac tcg tac tat gtg cac cag aat gcc aag cac agc 1915 Cys Ala Lys ArgAsn Ser Tyr Tyr Val His Gln Asn Ala Lys His Ser 555 560 565 tgg gtg ccctac gag cct gac gat gac gcc cag aag tgt gag ctg atc 1963 Trp Val Pro TyrGlu Pro Asp Asp Asp Ala Gln Lys Cys Glu Leu Ile 570 575 580 tgc cag tcggcg gac aca ggg gac gtg gtg ttc atg aac cag gtg gtt 2011 Cys Gln Ser AlaAsp Thr Gly Asp Val Val Phe Met Asn Gln Val Val 585 590 595 cac gat gggaca cgc tgc agc tac cgg gac cca tac agc gtc tgt gcg 2059 His Asp Gly ThrArg Cys Ser Tyr Arg Asp Pro Tyr Ser Val Cys Ala 600 605 610 cgt ggc gagtgt gtg cct gtc ggc tgt gac aag gag gtg ggg tcc atg 2107 Arg Gly Glu CysVal Pro Val Gly Cys Asp Lys Glu Val Gly Ser Met 615 620 625 630 aag gcggat gac aag tgt gga gtc tgc ggg ggt gac aac tcc cac tgc 2155 Lys Ala AspAsp Lys Cys Gly Val Cys Gly Gly Asp Asn Ser His Cys 635 640 645 agg actgtg aag ggg acg ctg ggc aag gcc tcc aag cag gca gga gct 2203 Arg Thr ValLys Gly Thr Leu Gly Lys Ala Ser Lys Gln Ala Gly Ala 650 655 660 ctc aagctg gtg cag atc cca gca ggt gcc agg cac atc cag att gag 2251 Leu Lys LeuVal Gln Ile Pro Ala Gly Ala Arg His Ile Gln Ile Glu 665 670 675 gca ctggag aag tcc ccc cac cgc att gtg gtg aag aac cag gtc acc 2299 Ala Leu GluLys Ser Pro His Arg Ile Val Val Lys Asn Gln Val Thr 680 685 690 ggc agcttc atc ctc aac ccc aag ggc aag gaa gcc aca agc cgg acc 2347 Gly Ser PheIle Leu Asn Pro Lys Gly Lys Glu Ala Thr Ser Arg Thr 695 700 705 710 ttcacc gcc atg ggc ctg gag tgg gag gat gcg gtg gag gat gcc aag 2395 Phe ThrAla Met Gly Leu Glu Trp Glu Asp Ala Val Glu Asp Ala Lys 715 720 725 gaaagc ttc aag acc agc ggg ccc ctg cct gaa gcc att gcc atc ctg 2443 Glu SerPhe Lys Thr Ser Gly Pro Leu Pro Glu Ala Ile Ala Ile Leu 730 735 740 gctctc ccc cca act gag ggt ggc ccc cgc agc agc ctg gcc tac aag 2491 Ala LeuPro Pro Thr Glu Gly Gly Pro Arg Ser Ser Leu Ala Tyr Lys 745 750 755 tacgtc atc cat gag gac ctg ctg ccc ctt atc ggg agc aac aat gtg 2539 Tyr ValIle His Glu Asp Leu Leu Pro Leu Ile Gly Ser Asn Asn Val 760 765 770 ctcctg gag gag atg gac acc tat gag tgg gcg ctc aag agc tgg gcc 2587 Leu LeuGlu Glu Met Asp Thr Tyr Glu Trp Ala Leu Lys Ser Trp Ala 775 780 785 790ccc tgc agc aag gcc tgt gga gga ggg atc cag ttc acc aaa tac ggc 2635 ProCys Ser Lys Ala Cys Gly Gly Gly Ile Gln Phe Thr Lys Tyr Gly 795 800 805tgc cgg cgc aga cga gac cac cac atg gtg cag cga cac ctg tgt gac 2683 CysArg Arg Arg Arg Asp His His Met Val Gln Arg His Leu Cys Asp 810 815 820cac aag aag agg ccc aag ccc atc cgc cgg cgc tgc aac cag cac ccg 2731 HisLys Lys Arg Pro Lys Pro Ile Arg Arg Arg Cys Asn Gln His Pro 825 830 835tgc tct cag cct gtg tgg gtg acg gag gag tgg ggt gcc tgc agc cgg 2779 CysSer Gln Pro Val Trp Val Thr Glu Glu Trp Gly Ala Cys Ser Arg 840 845 850agc tgt ggg aag ctg ggg gtg cag aca cgg ggg ata cag tgc ctg atg 2827 SerCys Gly Lys Leu Gly Val Gln Thr Arg Gly Ile Gln Cys Leu Met 855 860 865870 ccc ctc tcc aat gga acc cac aag gtc atg ccg gcc aaa gcc tgt gcc 2875Pro Leu Ser Asn Gly Thr His Lys Val Met Pro Ala Lys Ala Cys Ala 875 880885 ggg gac cgg cct gag gcc cga cgg ccc tgt ctc cga gtg ccc tgc cca 2923Gly Asp Arg Pro Glu Ala Arg Arg Pro Cys Leu Arg Val Pro Cys Pro 890 895900 gcc cag tgg agg ctg gga gcc tgg tcc cag tgc tct gcc acc tgt gga 2971Ala Gln Trp Arg Leu Gly Ala Trp Ser Gln Cys Ser Ala Thr Cys Gly 905 910915 gag ggc atc cag cag cgg cag gtg gtg tgc agg acc aac gcc aac agc 3019Glu Gly Ile Gln Gln Arg Gln Val Val Cys Arg Thr Asn Ala Asn Ser 920 925930 ctc ggg cat tgc gag ggg gat agg cca gac act gtc cag gtc tgc agc 3067Leu Gly His Cys Glu Gly Asp Arg Pro Asp Thr Val Gln Val Cys Ser 935 940945 950 ctg ccc gcc tgt gga gga aat cac cag aac tcc acg gtg agg gcc gat3115 Leu Pro Ala Cys Gly Gly Asn His Gln Asn Ser Thr Val Arg Ala Asp 955960 965 gtc tgg gaa ctt ggg acg cca gag ggg cag tgg gtg cca caa tct gaa3163 Val Trp Glu Leu Gly Thr Pro Glu Gly Gln Trp Val Pro Gln Ser Glu 970975 980 ccc cta cat ccc att aac aag ata tca tca acg gag ccc tgc acg gga3211 Pro Leu His Pro Ile Asn Lys Ile Ser Ser Thr Glu Pro Cys Thr Gly 985990 995 gac agg tct gtc ttc tgc cag atg gaa gtg ctc gat cgc tac tgc tcc3259 Asp Arg Ser Val Phe Cys Gln Met Glu Val Leu Asp Arg Tyr Cys Ser1000 1005 1010 att ccc ggc tac cac cgg ctc tgc tgt gtg tcc tgc atc aagaag gcc 3307 Ile Pro Gly Tyr His Arg Leu Cys Cys Val Ser Cys Ile Lys LysAla 1015 1020 1025 1030 tcg ggc ccc aac cct ggc cca gac cct ggc cca acctca ctg ccc ccc 3355 Ser Gly Pro Asn Pro Gly Pro Asp Pro Gly Pro Thr SerLeu Pro Pro 1035 1040 1045 ttc tcc act cct gga agc ccc tta cca gga ccccag gac cct gca gat 3403 Phe Ser Thr Pro Gly Ser Pro Leu Pro Gly Pro GlnAsp Pro Ala Asp 1050 1055 1060 gct gca gag cct cct gga aag cca acg ggatca gag gac cat cag cat 3451 Ala Ala Glu Pro Pro Gly Lys Pro Thr Gly SerGlu Asp His Gln His 1065 1070 1075 ggc cga gcc aca cag ctc cca gga gctctg gat aca agc tcc cca ggg 3499 Gly Arg Ala Thr Gln Leu Pro Gly Ala LeuAsp Thr Ser Ser Pro Gly 1080 1085 1090 acc cag cat ccc ttt gcc cct gagaca cca atc cct gga gca tcc tgg 3547 Thr Gln His Pro Phe Ala Pro Glu ThrPro Ile Pro Gly Ala Ser Trp 1095 1100 1105 1110 agc atc tcc cct acc accccc ggg ggg ctg cct tgg ggc tgg act cag 3595 Ser Ile Ser Pro Thr Thr ProGly Gly Leu Pro Trp Gly Trp Thr Gln 1115 1120 1125 aca cct acg cca gtccct gag gac aaa ggg caa cct gga gaa gac ctg 3643 Thr Pro Thr Pro Val ProGlu Asp Lys Gly Gln Pro Gly Glu Asp Leu 1130 1135 1140 aga cat ccc ggcacc agc ctc cct gct gcc tcc ccg gtg aca tga 3688 Arg His Pro Gly Thr SerLeu Pro Ala Ala Ser Pro Val Thr * 1145 1150 1155 gctgtgccct gccatcccactggcacgttt acactctgtg tactgccccg tgactcccag 3748 ctcagaggac acacatagcagggcaggcgc aagcacagac ttcattttaa atcattcgcc 3808 ttcttctcgt ttggggctgtgatgctcttt accccacaaa gcggggtggg aggaagacaa 3868 agatcaggga aagccctaatcggagatacc tcagcaagct gcccccggcg ggactgaccc 3928 tctcagggcc cctgttggtctcccctgcca agaccagggt caactattgc tccctcctca 3988 cagaccctgg gcctgggcagatctgaatcc cggctggtct gtagctagaa gctgtcaggg 4048 ctgcctgcct tcccggaactgtgaggaccc ctgtggaggc cctgcatatt tggcccctct 4108 ccccagaaag gcaaagcagggccagggtag gtgggggact gttcacagcc aggccgagag 4168 gaggggggcc tgggaatgtggcatgaggct tcccagctgc agggctggag ggggtggaac 4228 acaagatgat cgcaggcccagctcccggaa gccaagagct ccatgcagtt ccaccagctg 4288 aggccaggca gcagaggccagtttgtcttt gctggccaga agatggtgct catggccata 4348 ctctggcctt gcagatgtcactagtgttac ttctagtgag tccagattac agactggccc 4408 cccaatctca ccccagcccaccagagaagg gggctcagga caccctggac cccaagtcct 4468 cagcatccag ggatttccaaactggcgctc accccctgac tccaccagga tggcaacttc 4528 aattatcact ctcagcctggaaggggactc tgtgggacac agagggaaca cgatttctca 4588 ggctgtccct tcaatcattgcccttctccg aagatcgctc ctgctggagt cggacatctt 4648 catcttctac ctggctcaagctgggccaga gtgtgtggtt ctcccagggg tggttggacc 4708 ccaggactga ggaccagagtctactcatag cctggccctg gagatgacaa gggccaccca 4768 ggccaagtgc cccagggcagggtgccagcc cctggcctgg tgctggagtg gggaagacac 4828 actcacccac ggtgctgtaagggcctgagc tgtgctcagc tgccggccat gctacctcca 4888 agggacaggt aacagtcttagatcctctgg ctctcaggaa gtggcagggg gtcccaggac 4948 acctccgggg tcttggaggatgtctcctaa actcctgcca ggtgtagagg tgcttctcac 5008 ttcttccttc cccaaggcaaaggggctgtt ctgagccagc ctggaggaac atgagtagtg 5068 ggcccctggc ctgcaacccctttggagagt ggaggtcctg gggggctccc cgccctcccc 5128 ctgttgccct cccctccctgggatgctggg gcacacgtgg agtcattcct gtgagaacca 5188 gcctggcctg tgttaaactcttgtgccttg gaaatccaga tctttaaaat tttatgtatt 5248 tattaacatc gccattgggccccaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5308 aaaaaaaaaa aaaaaaaaaaaaaaaggggc ggccgc 5344 5 1156 PRT Homo sapiens 5 Met Val Val Asp Thr ProPro Thr Leu Pro Arg His Ser Ser His Leu 1 5 10 15 Arg Val Ala Arg SerPro Leu His Pro Gly Gly Thr Leu Trp Pro Gly 20 25 30 Arg Val Gly Arg HisSer Leu Tyr Phe Asn Val Thr Val Phe Gly Lys 35 40 45 Glu Leu His Leu ArgLeu Arg Pro Asn Arg Arg Leu Val Val Pro Gly 50 55 60 Ser Ser Val Glu TrpGln Glu Asp Phe Arg Glu Leu Phe Arg Gln Pro 65 70 75 80 Leu Arg Gln GluCys Val Tyr Thr Gly Gly Val Thr Gly Met Pro Gly 85 90 95 Ala Ala Val AlaIle Ser Asn Cys Asp Gly Leu Ala Gly Leu Ile Arg 100 105 110 Thr Asp SerThr Asp Phe Phe Ile Glu Pro Leu Glu Arg Gly Gln Gln 115 120 125 Glu LysGlu Ala Ser Gly Arg Thr His Val Val Tyr Arg Arg Glu Ala 130 135 140 ValGln Gln Glu Trp Ala Glu Pro Asp Gly Asp Leu His Asn Glu Ala 145 150 155160 Phe Gly Leu Gly Asp Leu Pro Asn Leu Leu Gly Leu Val Gly Asp Gln 165170 175 Leu Gly Asp Thr Glu Arg Lys Arg Arg His Ala Lys Pro Gly Ser Tyr180 185 190 Ser Ile Glu Val Leu Leu Val Val Asp Asp Ser Val Val Arg PheHis 195 200 205 Gly Lys Glu His Val Gln Asn Tyr Val Leu Thr Leu Met AsnIle Val 210 215 220 Asp Glu Ile Tyr His Asp Glu Ser Leu Gly Val His IleAsn Ile Ala 225 230 235 240 Leu Val Arg Leu Ile Met Val Gly Tyr Arg GlnSer Leu Ser Leu Ile 245 250 255 Glu Arg Gly Asn Pro Ser Arg Ser Leu GluGln Val Cys Arg Trp Ala 260 265 270 His Ser Gln Gln Arg Gln Asp Pro SerHis Ala Glu His His Asp His 275 280 285 Val Val Phe Leu Thr Arg Gln AspPhe Gly Pro Ser Gly Tyr Ala Pro 290 295 300 Val Thr Gly Met Cys His ProLeu Arg Ser Cys Ala Leu Asn His Glu 305 310 315 320 Asp Gly Phe Ser SerAla Phe Val Ile Ala His Glu Thr Gly His Val 325 330 335 Leu Gly Met GluHis Asp Gly Gln Gly Asn Gly Cys Ala Asp Glu Thr 340 345 350 Ser Leu GlySer Val Met Ala Pro Leu Val Gln Ala Ala Phe His Arg 355 360 365 Phe HisTrp Ser Arg Cys Ser Lys Leu Glu Leu Ser Arg Tyr Leu Pro 370 375 380 SerTyr Asp Cys Leu Leu Asp Asp Pro Phe Asp Pro Ala Trp Pro Gln 385 390 395400 Pro Pro Glu Leu Pro Gly Ile Asn Tyr Ser Met Asp Glu Gln Cys Arg 405410 415 Phe Asp Phe Gly Ser Gly Tyr Gln Thr Cys Leu Ala Ile Arg Thr Phe420 425 430 Glu Pro Cys Lys Gln Leu Trp Cys Ser His Pro Asp Asn Pro TyrPhe 435 440 445 Cys Lys Thr Lys Lys Gly Pro Pro Leu Asp Gly Thr Glu CysAla Pro 450 455 460 Gly Lys Trp Cys Phe Lys Gly His Cys Ile Trp Lys SerPro Glu Gln 465 470 475 480 Thr Tyr Gly Gln Asp Gly Gly Trp Ser Ser TrpThr Lys Phe Gly Ser 485 490 495 Cys Ser Arg Ser Cys Gly Gly Gly Val ArgSer Arg Ser Arg Ser Cys 500 505 510 Asn Asn Pro Ser Pro Ala Tyr Gly GlyArg Pro Cys Leu Gly Pro Met 515 520 525 Phe Glu Tyr Gln Val Cys Asn SerGlu Glu Cys Pro Gly Thr Tyr Glu 530 535 540 Asp Phe Arg Ala Gln Gln CysAla Lys Arg Asn Ser Tyr Tyr Val His 545 550 555 560 Gln Asn Ala Lys HisSer Trp Val Pro Tyr Glu Pro Asp Asp Asp Ala 565 570 575 Gln Lys Cys GluLeu Ile Cys Gln Ser Ala Asp Thr Gly Asp Val Val 580 585 590 Phe Met AsnGln Val Val His Asp Gly Thr Arg Cys Ser Tyr Arg Asp 595 600 605 Pro TyrSer Val Cys Ala Arg Gly Glu Cys Val Pro Val Gly Cys Asp 610 615 620 LysGlu Val Gly Ser Met Lys Ala Asp Asp Lys Cys Gly Val Cys Gly 625 630 635640 Gly Asp Asn Ser His Cys Arg Thr Val Lys Gly Thr Leu Gly Lys Ala 645650 655 Ser Lys Gln Ala Gly Ala Leu Lys Leu Val Gln Ile Pro Ala Gly Ala660 665 670 Arg His Ile Gln Ile Glu Ala Leu Glu Lys Ser Pro His Arg IleVal 675 680 685 Val Lys Asn Gln Val Thr Gly Ser Phe Ile Leu Asn Pro LysGly Lys 690 695 700 Glu Ala Thr Ser Arg Thr Phe Thr Ala Met Gly Leu GluTrp Glu Asp 705 710 715 720 Ala Val Glu Asp Ala Lys Glu Ser Phe Lys ThrSer Gly Pro Leu Pro 725 730 735 Glu Ala Ile Ala Ile Leu Ala Leu Pro ProThr Glu Gly Gly Pro Arg 740 745 750 Ser Ser Leu Ala Tyr Lys Tyr Val IleHis Glu Asp Leu Leu Pro Leu 755 760 765 Ile Gly Ser Asn Asn Val Leu LeuGlu Glu Met Asp Thr Tyr Glu Trp 770 775 780 Ala Leu Lys Ser Trp Ala ProCys Ser Lys Ala Cys Gly Gly Gly Ile 785 790 795 800 Gln Phe Thr Lys TyrGly Cys Arg Arg Arg Arg Asp His His Met Val 805 810 815 Gln Arg His LeuCys Asp His Lys Lys Arg Pro Lys Pro Ile Arg Arg 820 825 830 Arg Cys AsnGln His Pro Cys Ser Gln Pro Val Trp Val Thr Glu Glu 835 840 845 Trp GlyAla Cys Ser Arg Ser Cys Gly Lys Leu Gly Val Gln Thr Arg 850 855 860 GlyIle Gln Cys Leu Met Pro Leu Ser Asn Gly Thr His Lys Val Met 865 870 875880 Pro Ala Lys Ala Cys Ala Gly Asp Arg Pro Glu Ala Arg Arg Pro Cys 885890 895 Leu Arg Val Pro Cys Pro Ala Gln Trp Arg Leu Gly Ala Trp Ser Gln900 905 910 Cys Ser Ala Thr Cys Gly Glu Gly Ile Gln Gln Arg Gln Val ValCys 915 920 925 Arg Thr Asn Ala Asn Ser Leu Gly His Cys Glu Gly Asp ArgPro Asp 930 935 940 Thr Val Gln Val Cys Ser Leu Pro Ala Cys Gly Gly AsnHis Gln Asn 945 950 955 960 Ser Thr Val Arg Ala Asp Val Trp Glu Leu GlyThr Pro Glu Gly Gln 965 970 975 Trp Val Pro Gln Ser Glu Pro Leu His ProIle Asn Lys Ile Ser Ser 980 985 990 Thr Glu Pro Cys Thr Gly Asp Arg SerVal Phe Cys Gln Met Glu Val 995 1000 1005 Leu Asp Arg Tyr Cys Ser IlePro Gly Tyr His Arg Leu Cys Cys Val 1010 1015 1020 Ser Cys Ile Lys LysAla Ser Gly Pro Asn Pro Gly Pro Asp Pro Gly 1025 1030 1035 1040 Pro ThrSer Leu Pro Pro Phe Ser Thr Pro Gly Ser Pro Leu Pro Gly 1045 1050 1055Pro Gln Asp Pro Ala Asp Ala Ala Glu Pro Pro Gly Lys Pro Thr Gly 10601065 1070 Ser Glu Asp His Gln His Gly Arg Ala Thr Gln Leu Pro Gly AlaLeu 1075 1080 1085 Asp Thr Ser Ser Pro Gly Thr Gln His Pro Phe Ala ProGlu Thr Pro 1090 1095 1100 Ile Pro Gly Ala Ser Trp Ser Ile Ser Pro ThrThr Pro Gly Gly Leu 1105 1110 1115 1120 Pro Trp Gly Trp Thr Gln Thr ProThr Pro Val Pro Glu Asp Lys Gly 1125 1130 1135 Gln Pro Gly Glu Asp LeuArg His Pro Gly Thr Ser Leu Pro Ala Ala 1140 1145 1150 Ser Pro Val Thr1155 6 3471 DNA Homo sapiens CDS (1)...(3471) 6 atg gta gtg gac acg ccaccc aca cta cca cga cac tcc agt cac ctc 48 Met Val Val Asp Thr Pro ProThr Leu Pro Arg His Ser Ser His Leu 1 5 10 15 cgg gtg gct cgc agc cctctg cac cca gga ggg acc ctg tgg cct ggc 96 Arg Val Ala Arg Ser Pro LeuHis Pro Gly Gly Thr Leu Trp Pro Gly 20 25 30 agg gtg ggg cgc cac tcc ctctac ttc aat gtc act gtt ttc ggg aag 144 Arg Val Gly Arg His Ser Leu TyrPhe Asn Val Thr Val Phe Gly Lys 35 40 45 gaa ctg cac ttg cgc ctg cgg cccaat cgg agg ttg gta gtg cca gga 192 Glu Leu His Leu Arg Leu Arg Pro AsnArg Arg Leu Val Val Pro Gly 50 55 60 tcc tca gtg gag tgg cag gag gat tttcgg gag ctg ttc cgg cag ccc 240 Ser Ser Val Glu Trp Gln Glu Asp Phe ArgGlu Leu Phe Arg Gln Pro 65 70 75 80 tta cgg cag gag tgt gtg tac act ggaggt gtc act gga atg cct ggg 288 Leu Arg Gln Glu Cys Val Tyr Thr Gly GlyVal Thr Gly Met Pro Gly 85 90 95 gca gct gtt gcc atc agc aac tgt gac ggattg gcg ggc ctc atc cgc 336 Ala Ala Val Ala Ile Ser Asn Cys Asp Gly LeuAla Gly Leu Ile Arg 100 105 110 aca gac agc acc gac ttc ttc att gag cctctg gag cgg ggc cag cag 384 Thr Asp Ser Thr Asp Phe Phe Ile Glu Pro LeuGlu Arg Gly Gln Gln 115 120 125 gag aag gag gcc agc ggg agg aca cat gtggtg tac cgc cgg gag gcc 432 Glu Lys Glu Ala Ser Gly Arg Thr His Val ValTyr Arg Arg Glu Ala 130 135 140 gtc cag cag gag tgg gca gaa cct gac ggggac ctg cac aat gaa gcc 480 Val Gln Gln Glu Trp Ala Glu Pro Asp Gly AspLeu His Asn Glu Ala 145 150 155 160 ttt ggc ctg gga gac ctt ccc aac ctgctg ggc ctg gtg ggg gac cag 528 Phe Gly Leu Gly Asp Leu Pro Asn Leu LeuGly Leu Val Gly Asp Gln 165 170 175 ctg ggc gac aca gag cgg aag cgg cggcat gcc aag cca ggc agc tac 576 Leu Gly Asp Thr Glu Arg Lys Arg Arg HisAla Lys Pro Gly Ser Tyr 180 185 190 agc atc gag gtg ctg ctg gtg gtg gacgac tcg gtg gtt cgc ttc cat 624 Ser Ile Glu Val Leu Leu Val Val Asp AspSer Val Val Arg Phe His 195 200 205 ggc aag gag cat gtg cag aac tat gtcctc acc ctc atg aat atc gta 672 Gly Lys Glu His Val Gln Asn Tyr Val LeuThr Leu Met Asn Ile Val 210 215 220 gat gag att tac cac gat gag tcc ctgggg gtt cat ata aat att gcc 720 Asp Glu Ile Tyr His Asp Glu Ser Leu GlyVal His Ile Asn Ile Ala 225 230 235 240 ctc gtc cgc ttg atc atg gtt ggctac cga cag tcc ctg agc ctg atc 768 Leu Val Arg Leu Ile Met Val Gly TyrArg Gln Ser Leu Ser Leu Ile 245 250 255 gag cgc ggg aac ccc tca cgc agcctg gag cag gtg tgt cgc tgg gca 816 Glu Arg Gly Asn Pro Ser Arg Ser LeuGlu Gln Val Cys Arg Trp Ala 260 265 270 cac tcc cag cag cgc cag gac cccagc cac gct gag cac cat gac cac 864 His Ser Gln Gln Arg Gln Asp Pro SerHis Ala Glu His His Asp His 275 280 285 gtt gtg ttc ctc acc cgg cag gacttt ggg ccc tca ggg tat gca ccc 912 Val Val Phe Leu Thr Arg Gln Asp PheGly Pro Ser Gly Tyr Ala Pro 290 295 300 gtc act ggc atg tgt cac ccc ctgagg agc tgt gcc ctc aac cat gag 960 Val Thr Gly Met Cys His Pro Leu ArgSer Cys Ala Leu Asn His Glu 305 310 315 320 gat ggc ttc tcc tca gcc ttcgtg ata gct cat gag acc ggc cac gtg 1008 Asp Gly Phe Ser Ser Ala Phe ValIle Ala His Glu Thr Gly His Val 325 330 335 ctc ggc atg gag cat gac ggtcag ggg aat ggc tgt gca gat gag acc 1056 Leu Gly Met Glu His Asp Gly GlnGly Asn Gly Cys Ala Asp Glu Thr 340 345 350 agc ctg ggc agc gtc atg gcgccc ctg gtg cag gct gcc ttc cac cgc 1104 Ser Leu Gly Ser Val Met Ala ProLeu Val Gln Ala Ala Phe His Arg 355 360 365 ttc cat tgg tcc cgc tgc agcaag ctg gag ctc agc cgc tac ctc ccc 1152 Phe His Trp Ser Arg Cys Ser LysLeu Glu Leu Ser Arg Tyr Leu Pro 370 375 380 tcc tac gac tgc ctc ctc gatgac ccc ttt gat cct gcc tgg ccc cag 1200 Ser Tyr Asp Cys Leu Leu Asp AspPro Phe Asp Pro Ala Trp Pro Gln 385 390 395 400 ccc cca gag ctg cct gggatc aac tac tca atg gat gag cag tgc cgc 1248 Pro Pro Glu Leu Pro Gly IleAsn Tyr Ser Met Asp Glu Gln Cys Arg 405 410 415 ttt gac ttt ggc agt ggctac cag acc tgc ttg gca atc agg acc ttt 1296 Phe Asp Phe Gly Ser Gly TyrGln Thr Cys Leu Ala Ile Arg Thr Phe 420 425 430 gag ccc tgc aag cag ctgtgg tgc agc cat cct gac aac ccg tac ttc 1344 Glu Pro Cys Lys Gln Leu TrpCys Ser His Pro Asp Asn Pro Tyr Phe 435 440 445 tgc aag acc aag aag gggccc ccg ctg gat ggg act gag tgt gca ccc 1392 Cys Lys Thr Lys Lys Gly ProPro Leu Asp Gly Thr Glu Cys Ala Pro 450 455 460 ggc aag tgg tgc ttc aaaggt cac tgc atc tgg aag tcg ccg gag cag 1440 Gly Lys Trp Cys Phe Lys GlyHis Cys Ile Trp Lys Ser Pro Glu Gln 465 470 475 480 aca tat ggc cag gatgga ggc tgg agc tcc tgg acc aag ttt ggg tca 1488 Thr Tyr Gly Gln Asp GlyGly Trp Ser Ser Trp Thr Lys Phe Gly Ser 485 490 495 tgt tcg cgg tca tgtggg ggc ggg gtg cga tcc cgc agc cgg agc tgc 1536 Cys Ser Arg Ser Cys GlyGly Gly Val Arg Ser Arg Ser Arg Ser Cys 500 505 510 aac aac ccc tcc ccagcc tat gga ggc cgc ccg tgc tta ggg ccc atg 1584 Asn Asn Pro Ser Pro AlaTyr Gly Gly Arg Pro Cys Leu Gly Pro Met 515 520 525 ttc gag tac cag gtctgc aac agc gag gag tgc cct ggg acc tac gag 1632 Phe Glu Tyr Gln Val CysAsn Ser Glu Glu Cys Pro Gly Thr Tyr Glu 530 535 540 gac ttc cgg gcc cagcag tgt gcc aag cgc aac tcg tac tat gtg cac 1680 Asp Phe Arg Ala Gln GlnCys Ala Lys Arg Asn Ser Tyr Tyr Val His 545 550 555 560 cag aat gcc aagcac agc tgg gtg ccc tac gag cct gac gat gac gcc 1728 Gln Asn Ala Lys HisSer Trp Val Pro Tyr Glu Pro Asp Asp Asp Ala 565 570 575 cag aag tgt gagctg atc tgc cag tcg gcg gac aca ggg gac gtg gtg 1776 Gln Lys Cys Glu LeuIle Cys Gln Ser Ala Asp Thr Gly Asp Val Val 580 585 590 ttc atg aac caggtg gtt cac gat ggg aca cgc tgc agc tac cgg gac 1824 Phe Met Asn Gln ValVal His Asp Gly Thr Arg Cys Ser Tyr Arg Asp 595 600 605 cca tac agc gtctgt gcg cgt ggc gag tgt gtg cct gtc ggc tgt gac 1872 Pro Tyr Ser Val CysAla Arg Gly Glu Cys Val Pro Val Gly Cys Asp 610 615 620 aag gag gtg gggtcc atg aag gcg gat gac aag tgt gga gtc tgc ggg 1920 Lys Glu Val Gly SerMet Lys Ala Asp Asp Lys Cys Gly Val Cys Gly 625 630 635 640 ggt gac aactcc cac tgc agg act gtg aag ggg acg ctg ggc aag gcc 1968 Gly Asp Asn SerHis Cys Arg Thr Val Lys Gly Thr Leu Gly Lys Ala 645 650 655 tcc aag caggca gga gct ctc aag ctg gtg cag atc cca gca ggt gcc 2016 Ser Lys Gln AlaGly Ala Leu Lys Leu Val Gln Ile Pro Ala Gly Ala 660 665 670 agg cac atccag att gag gca ctg gag aag tcc ccc cac cgc att gtg 2064 Arg His Ile GlnIle Glu Ala Leu Glu Lys Ser Pro His Arg Ile Val 675 680 685 gtg aag aaccag gtc acc ggc agc ttc atc ctc aac ccc aag ggc aag 2112 Val Lys Asn GlnVal Thr Gly Ser Phe Ile Leu Asn Pro Lys Gly Lys 690 695 700 gaa gcc acaagc cgg acc ttc acc gcc atg ggc ctg gag tgg gag gat 2160 Glu Ala Thr SerArg Thr Phe Thr Ala Met Gly Leu Glu Trp Glu Asp 705 710 715 720 gcg gtggag gat gcc aag gaa agc ttc aag acc agc ggg ccc ctg cct 2208 Ala Val GluAsp Ala Lys Glu Ser Phe Lys Thr Ser Gly Pro Leu Pro 725 730 735 gaa gccatt gcc atc ctg gct ctc ccc cca act gag ggt ggc ccc cgc 2256 Glu Ala IleAla Ile Leu Ala Leu Pro Pro Thr Glu Gly Gly Pro Arg 740 745 750 agc agcctg gcc tac aag tac gtc atc cat gag gac ctg ctg ccc ctt 2304 Ser Ser LeuAla Tyr Lys Tyr Val Ile His Glu Asp Leu Leu Pro Leu 755 760 765 atc gggagc aac aat gtg ctc ctg gag gag atg gac acc tat gag tgg 2352 Ile Gly SerAsn Asn Val Leu Leu Glu Glu Met Asp Thr Tyr Glu Trp 770 775 780 gcg ctcaag agc tgg gcc ccc tgc agc aag gcc tgt gga gga ggg atc 2400 Ala Leu LysSer Trp Ala Pro Cys Ser Lys Ala Cys Gly Gly Gly Ile 785 790 795 800 cagttc acc aaa tac ggc tgc cgg cgc aga cga gac cac cac atg gtg 2448 Gln PheThr Lys Tyr Gly Cys Arg Arg Arg Arg Asp His His Met Val 805 810 815 cagcga cac ctg tgt gac cac aag aag agg ccc aag ccc atc cgc cgg 2496 Gln ArgHis Leu Cys Asp His Lys Lys Arg Pro Lys Pro Ile Arg Arg 820 825 830 cgctgc aac cag cac ccg tgc tct cag cct gtg tgg gtg acg gag gag 2544 Arg CysAsn Gln His Pro Cys Ser Gln Pro Val Trp Val Thr Glu Glu 835 840 845 tggggt gcc tgc agc cgg agc tgt ggg aag ctg ggg gtg cag aca cgg 2592 Trp GlyAla Cys Ser Arg Ser Cys Gly Lys Leu Gly Val Gln Thr Arg 850 855 860 gggata cag tgc ctg atg ccc ctc tcc aat gga acc cac aag gtc atg 2640 Gly IleGln Cys Leu Met Pro Leu Ser Asn Gly Thr His Lys Val Met 865 870 875 880ccg gcc aaa gcc tgt gcc ggg gac cgg cct gag gcc cga cgg ccc tgt 2688 ProAla Lys Ala Cys Ala Gly Asp Arg Pro Glu Ala Arg Arg Pro Cys 885 890 895ctc cga gtg ccc tgc cca gcc cag tgg agg ctg gga gcc tgg tcc cag 2736 LeuArg Val Pro Cys Pro Ala Gln Trp Arg Leu Gly Ala Trp Ser Gln 900 905 910tgc tct gcc acc tgt gga gag ggc atc cag cag cgg cag gtg gtg tgc 2784 CysSer Ala Thr Cys Gly Glu Gly Ile Gln Gln Arg Gln Val Val Cys 915 920 925agg acc aac gcc aac agc ctc ggg cat tgc gag ggg gat agg cca gac 2832 ArgThr Asn Ala Asn Ser Leu Gly His Cys Glu Gly Asp Arg Pro Asp 930 935 940act gtc cag gtc tgc agc ctg ccc gcc tgt gga gga aat cac cag aac 2880 ThrVal Gln Val Cys Ser Leu Pro Ala Cys Gly Gly Asn His Gln Asn 945 950 955960 tcc acg gtg agg gcc gat gtc tgg gaa ctt ggg acg cca gag ggg cag 2928Ser Thr Val Arg Ala Asp Val Trp Glu Leu Gly Thr Pro Glu Gly Gln 965 970975 tgg gtg cca caa tct gaa ccc cta cat ccc att aac aag ata tca tca 2976Trp Val Pro Gln Ser Glu Pro Leu His Pro Ile Asn Lys Ile Ser Ser 980 985990 acg gag ccc tgc acg gga gac agg tct gtc ttc tgc cag atg gaa gtg 3024Thr Glu Pro Cys Thr Gly Asp Arg Ser Val Phe Cys Gln Met Glu Val 995 10001005 ctc gat cgc tac tgc tcc att ccc ggc tac cac cgg ctc tgc tgt gtg3072 Leu Asp Arg Tyr Cys Ser Ile Pro Gly Tyr His Arg Leu Cys Cys Val1010 1015 1020 tcc tgc atc aag aag gcc tcg ggc ccc aac cct ggc cca gaccct ggc 3120 Ser Cys Ile Lys Lys Ala Ser Gly Pro Asn Pro Gly Pro Asp ProGly 1025 1030 1035 1040 cca acc tca ctg ccc ccc ttc tcc act cct gga agcccc tta cca gga 3168 Pro Thr Ser Leu Pro Pro Phe Ser Thr Pro Gly Ser ProLeu Pro Gly 1045 1050 1055 ccc cag gac cct gca gat gct gca gag cct cctgga aag cca acg gga 3216 Pro Gln Asp Pro Ala Asp Ala Ala Glu Pro Pro GlyLys Pro Thr Gly 1060 1065 1070 tca gag gac cat cag cat ggc cga gcc acacag ctc cca gga gct ctg 3264 Ser Glu Asp His Gln His Gly Arg Ala Thr GlnLeu Pro Gly Ala Leu 1075 1080 1085 gat aca agc tcc cca ggg acc cag catccc ttt gcc cct gag aca cca 3312 Asp Thr Ser Ser Pro Gly Thr Gln His ProPhe Ala Pro Glu Thr Pro 1090 1095 1100 atc cct gga gca tcc tgg agc atctcc cct acc acc ccc ggg ggg ctg 3360 Ile Pro Gly Ala Ser Trp Ser Ile SerPro Thr Thr Pro Gly Gly Leu 1105 1110 1115 1120 cct tgg ggc tgg act cagaca cct acg cca gtc cct gag gac aaa ggg 3408 Pro Trp Gly Trp Thr Gln ThrPro Thr Pro Val Pro Glu Asp Lys Gly 1125 1130 1135 caa cct gga gaa gacctg aga cat ccc ggc acc agc ctc cct gct gcc 3456 Gln Pro Gly Glu Asp LeuArg His Pro Gly Thr Ser Leu Pro Ala Ala 1140 1145 1150 tcc ccg gtg acatga 3471 Ser Pro Val Thr * 1155 7 119 PRT Artificial Sequence consensus7 His Leu Glu Lys Asn Arg Ser Leu Leu Ala Pro Asp Phe Thr Val Thr 1 5 1015 Thr Tyr Asp Glu Asp Gly Thr Leu Val Thr Glu Glu Pro Leu Ile Gln 20 2530 Asp Asp His Cys Tyr Tyr Gln Gly Tyr Val Glu Gly Tyr Pro Asn Ser 35 4045 Ala Val Ser Leu Ser Thr Cys Ser Gly Gly Leu Arg Gly Ile Leu Gln 50 5560 Leu Glu Asn Leu Ser Tyr Gly Ile Glu Pro Leu Glu Ser Ser Asp Gly 65 7075 80 Phe Glu His Ile Ile Tyr Gln Ile Glu Asn Asp Lys Thr Glu Pro Ser 8590 95 Pro Cys Gly Glu Cys Gly Ser Leu Ser Thr Ser Thr Asp Ser Ser Tyr100 105 110 Gly Ile Arg Ser Ala Ser Pro 115 8 203 PRT ArtificialSequence consensus 8 Lys Tyr Ile Glu Leu Val Ile Val Val Asp His Gly MetTyr Thr Lys 1 5 10 15 Tyr Gly Ser Asp Leu Asn Lys Ile Arg Gln Arg ValHis Gln Ile Val 20 25 30 Asn Leu Val Asn Glu Ile Tyr Arg Pro Gln Leu AsnIle Arg Val Val 35 40 45 Leu Val Gly Leu Glu Ile Trp Ser Asp Gly Asp LysIle Asn Val Gln 50 55 60 Ser Asp Ala Asn Asp Thr Leu His Ser Phe Gly GluTrp Arg Glu Thr 65 70 75 80 Asp Leu Leu Lys Arg Lys Ser His Asp Asn AlaGln Leu Leu Thr Gly 85 90 95 Ile Asp Phe Asp Gly Asn Thr Ile Gly Ala AlaTyr Val Gly Gly Met 100 105 110 Cys Ser Pro Lys Arg Ser Val Gly Val ValGln Asp His Ser Pro Ile 115 120 125 Val Leu Leu Val Ala Val Thr Met AlaHis Glu Leu Gly His Asn Leu 130 135 140 Gly Met Thr His Asp Asp Lys AsnLys Asp Gly Cys Thr Cys Glu Gly 145 150 155 160 Gly Gly Ser Cys Ile MetAsn Pro Val Ala Ser Ser Ser Pro Ser Lys 165 170 175 Lys Lys Phe Ser AsnCys Ser Lys Asp Asp Tyr Gln Lys Phe Leu Thr 180 185 190 Lys Gln Lys ProGln Cys Leu Leu Asn Lys Pro 195 200 9 54 PRT Artificial Sequenceconsensus 9 Ser Pro Trp Ser Glu Trp Ser Pro Cys Ser Val Thr Cys Gly LysGly 1 5 10 15 Ile Arg Thr Arg Gln Arg Thr Cys Asn Ser Pro Ala Pro GlnLys Lys 20 25 30 Gly Gly Lys Pro Cys Thr Gly Asp Ala Gln Glu Glu Thr GluAla Cys 35 40 45 Asp Met Met Asp Lys Cys 50 10 22 PRT ArtificialSequence consensus 10 Leu Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa XaaXaa Xaa Leu Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Leu 20 11 10 PRTArtificial Sequence consensus 11 Xaa Xaa Xaa His Glu Xaa Xaa His Xaa Xaa1 5 10

What is claimed is:
 1. An isolated nucleic acid molecule selected fromthe group consisting of: a)a nucleic acid molecule comprising anucleotide sequence which is at least 80% identical to the nucleotidesequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, orthe cDNA insert of the plasmid deposited with the ATCC as AccessionNumber ______; b) a nucleic acid molecule comprising a fragment of atleast 450 nucleotides of the nucleotide sequence of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or the cDNA insert of the plasmiddeposited with the ATCC as Accession Number ______; c) a nucleic acidmolecule which encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO: 5, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession Number______; d) a nucleic acid molecule which encodes a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 5, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number ______, wherein thefragment comprises at least 15 contiguous amino acids of SEQ ID NO: 2,SEQ ID NO: 5, or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with the ATCC as Accession Number ; and e) anucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 5, or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Accession Number______, wherein the nucleic acid molecule hybridizes to a nucleic acidmolecule comprising SEQ ID NO: 1, 3, 4, 6, or a complement thereof,under stringent conditions.
 2. The isolated nucleic acid molecule ofclaim 1, which is selected from the group consisting of: a) a nucleicacid comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, or the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ; and b) a nucleic acid molecule whichencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO: 5, or the amino acid sequence encoded by the cDNA insertof the plasmid deposited with the ATCC as Accession Number ______. 3.The nucleic acid molecule of claim 1 further comprising vector nucleicacid sequences.
 4. The nucleic acid molecule of claim 1 furthercomprising nucleic acid sequences encoding a heterologous polypeptide.5. A host cell which contains the nucleic acid molecule of claim
 1. 6.The host cell of claim 5 which is a mammalian host cell.
 7. A non-humanmammalian host cell containing the nucleic acid molecule of claim
 1. 8.An isolated polypeptide selected from the group consisting of: a) apolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence which is at least 80% identical to a nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 6, the amino acid sequence encoded by the cDNA insertof the plasmid deposited with the ATCC as Accession Number ______, or acomplement thereof. b) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ IDNO: 5, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession Number ______, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, or a complement thereof under stringent conditions; andc) a fragment of a polypeptide comprising the amino acid sequence of SEQID NO: 2, SEQ ID NO: 5, or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Accession Number______, wherein the fragment comprises at least 15 contiguous aminoacids of SEQ ID NO: 2 or SEQ ID NO:
 5. 9. The isolated polypeptide ofclaim 8 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:5.
 10. The polypeptide of claim 8 further comprising heterologous aminoacid sequences.
 11. An antibody which selectively binds to a polypeptideof claim
 8. 12. A method for producing a polypeptide selected from thegroup consisting of: a) a polypeptide comprising the amino acid sequenceof SEQ ID NO: 2, SEQ ID NO: 5, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession Number______; b) a polypeptide comprising a fragment of the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 5, or the amino acid sequenceencoded by the cDNA insert of the plasmid deposited with the ATCC asAccession Number ______, wherein the fragment comprises at least 15contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 5, or the amino acidsequence encoded by the cDNA insert of the plasmid deposited with theATCC as Accession Number ______; and c) a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 5, or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Accession Number______, wherein the polypeptide is encoded by a nucleic acid moleculewhich hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, or a complement thereof understringent conditions; comprising culturing the host cell of claim 5under conditions in which the nucleic acid molecule is expressed.
 13. Amethod for detecting the presence of a polypeptide of claim 8 in asample, comprising: a) contacting the sample with a compound whichselectively binds to a polypeptide of claim 8; and b) determiningwhether the compound binds to the polypeptide in the sample.
 14. Themethod of claim 13, wherein the compound which binds to the polypeptideis an antibody.
 15. A kit comprising a compound which selectively bindsto a polypeptide of claim 8 and instructions for use.
 16. A method fordetecting the presence of a nucleic acid molecule of claim 1 in asample, comprising the steps of: a) contacting the sample with a nucleicacid probe or primer which selectively hybridizes to the nucleic acidmolecule; and b) determining whether the nucleic acid probe or primerbinds to a nucleic acid molecule in the sample.
 17. The method of claim16, wherein the sample comprises mRNA molecules and is contacted with anucleic acid probe.
 18. A kit comprising a compound which selectivelyhybridizes to a nucleic acid molecule of claim 1 and instructions foruse.
 19. A method for identifying a compound which binds to apolypeptide of claim 8 comprising the steps of: a) contacting apolypeptide, or a cell expressing a polypeptide of claim 8 with a testcompound; and b) determining whether the polypeptide binds to the testcompound.
 20. The method of claim 19, wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay for53014-mediated signal transduction.
 21. A method for modulating theactivity of a polypeptide of claim 8 comprising contacting a polypeptideor a cell expressing a polypeptide of claim 8 with a compound whichbinds to the polypeptide in a sufficient concentration to modulate theactivity of the polypeptide.
 22. A method for identifying a compoundwhich modulates the activity of a polypeptide of claim 8, comprising: a)contacting a polypeptide of claim 8 with a test compound; and b)determining the effect of the test compound on the activity of thepolypeptide to thereby identify a compound which modulates the activityof the polypeptide.